Viral vectors exhibiting improved gene delivery properties

ABSTRACT

The technology described herein provides viral capsid polypeptides bearing mutations that alters tissue tropism of a virus comprising the viral capsid polypeptide. In various embodiments, tissue tropisim to the heart, kidney, liver, lung, spleen, or blood is altered.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/US19/31307 filed May 8, 2019, which designates the U.S. and claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 62/671,949 filed May 15, 2018, the contents of which is incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under RM1 HG008525 and NHGRI CEGS CCV awarded by National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 31, 2019, is named 002806-092110WOPT_SL.txt and is 15,134 bytes in size.

TECHNICAL FIELD OF THE INVENTION

This invention is related to viral vectors with modified tropism.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is an attractive agent for use as gene delivery vector. Its simple structure also makes it an attractive target for genetic improvement programs. Nonetheless, there is a continuing need in the art to improve delivery of DNA using AAV and other viral vectors. In particular, there is a need in the art for viral vectors with modified tissue or cell tropism.

SUMMARY OF THE INVENTION

Described herein are viral vectors with modified tropism. Such vectors provide an improvement in the degree of tissue targeting attainable with such vectors. In particular, viral vectors with capsid polypeptide mutations that modify tropism of the viral particles relative to particles with wild-type capsid polypeptide are described. Through systematic mutation of viral capsid polypeptides, specific amino acid residues and amino acid regions have been identified that when mutated increase or decrease tropism of the virus for certain tissue or cell types. This provides the ability to increase the targeting of a given tissue or cell type by a viral vector, or, conversely, to decrease the targeting of a given tissue or cell type by a viral vector depending upon the site-specific mutation(s) introduced to the capsid polypeptide. Indeed, it has been found herein that not only can one improve the tropism of a viral vector by incorporating amino acid changes that increase tropism for a desired tissue, but nucleic acid delivery can also be improved to a given tissue or cell type by reducing the efficiency with which a viral vector infects tissues or cells other than the desired tissue or cell. Mutations that achieve both of these effects, and their use to improve delivery to desired or targeted tissues or cell types are described herein. Further, it has surprisingly been found that one can combine, within a single viral capsid polypeptide, mutations that improve tropism for a desired or targeted tissue or cell type with mutations that reduce tropism for a non-desired tissue or cell type, and thereby further improve targeting efficiency of the vector.

One aspect of the technology described herein provides a viral capsid polypeptide bearing a mutation relative to SEQ ID NO: 1 (WT AAV2) that alters tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 1-7.

In one embodiment of any aspect, the tissue is heart, kidney, liver, lung, spleen, or blood.

In one embodiment of any aspect, the tropism to the tissue is increased. In one embodiment of any aspect, the tropism to the tissue is decreased.

Another aspect of the technology described herein provides a viral capsid polypeptide bearing a mutation that corresponds to a mutation of the polypeptide of SEQ ID NO: 1, the mutation selected for the group consisting of the mutations in Tables 1-9 relative to SEQ ID NO: 1.

Yet another aspect of the technology described herein provides a viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO: 2, wherein the region corresponding to the amino acid sequence of SEQ ID NO: 2 comprises a mutation relative to SEQ ID NO: 2 that alters tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 10-15.

Another aspect of the technology described herein provides a nucleic acid encoding any of the the viral capsid polypeptides described herein.

Another aspect of the technology described herein provides a viral particle comprising any of the the viral capsid polypeptides described herein.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a cell, the method comprising; contacting the cell with a viral particle comprising any of the the viral capsid polypeptides described herein

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a blood cell, the method comprising; contacting a blood cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 1.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a heart cell, the method comprising; contacting a heart cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 2

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a kidney cell, the method comprising; contacting a kidney cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 3.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a liver cell, the method comprising; contacting a liver cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 4.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a lung cell, the method comprising; contacting a lung cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 5

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a spleen cell, the method comprising; contacting a spleen cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 6.

In one embodiment of any aspect, the delivering is at least 1.1-fold more efficient as compared to a wild-type viral capsid polypeptide.

Another aspect of the technology described herein provides a method of reducing tissue tropism of a virus comprising a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1, the method comprising introducing a mutation set out in any of Tables 7-9 (deenrichment Tables).

Another aspect of the technology described herein provides a method of increasing delivery of a nucleic acid to a cell of a kidney, heart, or lung, the method comprising; contacting a cell of a kidney, heart, or lung with a viral particle comprising a viral capsid polypeptide comprising a mutation that reduces delivery of a nucleic acid to a cell of a liver, blood, or spleen.

In one embodiment of any aspect, the mutation that reduces delivery of a nucleic acid to a cell of a liver, blood, or spleen is selected from any of Tables 7-9.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a lung cell, the method comprising; contacting a lung cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 5 and a mutation selected from any of Tables 7-9.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a heart cell, the method comprising; contacting a heart cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 2 and a mutation selected from any of Tables 7-9.

Another aspect of the technology described herein provides a method of delivering a nucleic acid to a kidney cell, the method comprising; contacting a kidney cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 3 and a mutation selected from any of Tables 7-9.

Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in the region of amino acids selected the group consisting of: 440-460 of SEQ ID NO: 1, 475-505 of SEQ ID NO: 1, 518-532 of SEQ ID NO: 1, and 560-590 of SEQ ID NO: 1 that alters tissue tropism of a virus comprising the viral capsid polypeptide.

Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the kidney, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of:

(SEQ ID NO: 3) DEEEIATTNPVATEQYGDVSENLMHFQN; (SEQ ID NO: 4) DEEEIRQTNPVATEGYGEVSTNLMHGNK; (SEQ ID NO: 5) DEEEIRTTNPVATEQYGIVnStTNLNEGNR; (SEQ ID NO: 6) DEEEIRTTNPVATECYGSVSTDLQSGNL; (SEQ ID NO: 7) DENEIRTTNPVATEIYGSVSTeNLQNnGdNR; (SEQ ID NO: 8) DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR; (SEQ ID NO: 9) DEEEIRTTNPVATEQYGDVSENLMHFQN.

Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the liver, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of:

(SEQ ID NO: 10) DEEEIRTTNPVATEQYGVVSDNLQRGNR; (SEQ ID NO: 11) DECEIRTTNPVATEQYGSVGENLQRGNR; (SEQ ID NO: 12) DEEEIRTTNPVATEQYGVVSENLQRGNR; (SEQ ID NO: 13) DESEITTTNPVATEQYGWVSTNQQRGNR; (SEQ ID NO: 14) HELEIATTNPVATEQYGSASTNIQRGNR; (SEQ ID NO: 15) DEEEIATTNPVATEQYGGVSTNLQRGNR.

Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the lung, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of:

(SEQ ID NO: 16) DEEEIVTTNPVATEQYGNVSTNLQRGNR; (SEQ ID NO: 17) DEDEISTTNPVATEQYGSCSTNLQRGNR; (SEQ ID NO: 18) QEEEIRTTNPVATEQYGSVSTNLQRGDR: (SEQ ID NO: 19) NEEEIRTTNPCATEVYGSVSTNLQRGNR; (SEQ ID NO: 20) DEQEIVTTNPVATEVYGTVSTNLQRGNR.

Another aspect of the technology described herein provides a method of altering tropism of a virus comprising a capsid polypeptide corresponding to the polypeptide of SEQ ID NO: 1, the method comprising introducing a mutation in at least 2 regions selected from the group consisting of: amino acids 440-460 of SEQ ID NO: 1, amino acids 475-505 of SEQ ID NO: 1, amino acids 518-532 of SEQ ID NO: 1, and amino acids 560-590 of SEQ ID NO: 1.

Another aspect provides a viral capsid polypeptide bearing a mutation relative to SEQ ID NO: 1 (WT AAV2) that alters packaging efficiency of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in Table 16.

Definitions

As used herein the term “alters tropism” refers to a mutation or set of mutations that changes the efficiency with which a viral vector delivers a nucleic acid to a given tissue or cell type. The tropism of a virus or viral vector is generally defined by the structure of its outer surface that interacts with receptors or other cell surface determinants on target cells. For AAV vectors, among others, viral vector tropism is determined primarily by viral capsid polypeptides, and as described herein, the tropism of such vectors can be changed by changing the amino acid sequence of the viral capsid polypeptide. An amino acid change that changes the efficiency of viral vector delivery of a nucleic acid to a target cell or tissue type by at least 10% relative to a reference vector, often, but not necessarily relative to a wild-type vector, is an altered tropism. To be clear, an altered tropism can be an increase/enrichment by at least 10% (1.1×) or a decrease/de-enrichment by at least 10% (0.9×).

As used herein, the term “increases tropism: or “increased tropism” refers to an increase in efficiency of viral vector delivery of a nucleic acid to a target cell or tissue type by at least 0.1-fold or 10% relative to a reference vector. In various embodiments, the increase in efficiency of viral vector delivery of a nucleic acid to a target cell or tissue is at least 1.1× (i.e., 10% greater), 1.2× (i.e., 20% greater), at least 1.5× (i.e., 50% greater), at least 2.0× (i.e., doubled), at least 5.0× or at least 10.0× greater relative to the reference vector.

As used herein, the term “decreases tropism” or “decreased tropism” refers to a decrease in efficiency of viral vector delivery of a nucleic acid to a target cell or tissue type by at least 10% relative to a reference vector. In various embodiments, the efficiency of viral vector delivery of a nucleic acid to a target cell or tissue is at most 0.9× (10% lower, or 90% of reference), at most 0.8× (20% lower, or 80% of reference), at most 0.7× (30% lower, or 70% of reference), at most 0.5× (50% lower, or 50% of reference), at most 0.3× (70% lower, or 30% of reference), at most 0.2× (80% lower, or 20% of reference), at most 0.1× (90% lower, or 10% of reference) or lower relative to the reference vector.

As used herein, the term “corresponding to,” when used in reference to an amino acid or polynucleotide sequence means that a given amino acid or polynucleotide sequence in one polypeptide or polynucleotide molecule has structural properties, functional properties, or both that are similar relative to an amino acid or polynucleotide sequence in a similar location in another polypeptide or polynucleotide molecule. Homologues of a given polypeptide in different species “correspond to” each other, as do regions or domains of homologous polypeptides from different species. Similarly, capsid polypeptides of different serotypes of viral vectors, including but not limited to adeno-associated virus (AAV) vectors, “correspond to” each other, as do regions of such polypeptides, defined, for example by alignment of their amino acid sequences. While other alignment parameters can be used to define such regions, for the avoidance of doubt, alignment can be performed using BLAST® (Basic Local Alignment Search Tool) using default parameters of version BLAST+ 2.8.0 released Mar. 28, 2018.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a heat map of all path numbers searched versus that distance from wild-type (e.g., the number of mutations relative to wild-type). Beneficial mutations, neutral mutations, and deleterious mutations are shown. Most mutations are deleterious.

FIG. 2 shows a heat map of a subset of path numbers searched versus that distance from wild-type (e.g., the number of mutations relative to wild-type. Beneficial mutations, neutral mutations, and deleterious mutations are shown. Heat map highlights that longer paths quickly die off.

FIG. 3 shows a chart plotting the fitness at first mutation versus the distance at which fitness drops below wild-type (e.g., when the virus is no longer viable). On the x-axis, 0.0 indicates to a neutral mutation (e.g., a mutation that does not alter fitness of the virus), a positive value indicates to a beneficial mutation (e.g., a mutation that increases the fitness of the virus), and a negative value indicates a deleterious mutation (e.g., a mutation that decreases the fitness of the virus). Values on the y-axis indicate the number of mutations relative to wild-type.

FIG. 4 is a line graph of path proportions showing at what mutation rate the mutation library fails. Random mutations result in a sharp decline at 1 mutation relative to wild-type, whereas fast (e.g., deep search mutations) mutations can tolerate up to 6 mutations. Deep search mutations are more than 40×more efficient that random search at distances greater than or equal to 6 mutations relative to wild-type.

FIG. 5 shows a heat map of amino acid insertions introduced into the AAV2 viral capsid polypeptide that are beneficial, neutral, or deleterious to viral packaging.

FIG. 6 shows a heat map of amino acid substitutions introduced into the AAV2 viral capsid polypeptide that are beneficial, neutral, or deleterious to viral packaging.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are viral vectors with modified tropism. Such vectors provide an improvement in the degree of tissue targeting attainable with such vectors. In particular, viral vectors with capsid polypeptide mutations that modify tropism of the viral particles relative to particles with wild-type capsid polypeptide are described. Through systematic mutation of viral capsid polypeptides, specific amino acid residues and amino acid regions have been identified that when mutated increase or decrease tropism of the virus for certain tissue or cell types. This provides the ability to increase the targeting of a given tissue or cell type by a viral vector, or, conversely, to decrease the targeting of a given tissue or cell type by a viral vector depending upon the site-specific mutation(s) introduced to the capsid polypeptide. Indeed, it has been found herein that not only can one improve the tropism of a viral vector by incorporating amino acid changes that increase tropism for a desired tissue, but nucleic acid delivery can also be improved to a given tissue or cell type by reducing the efficiency with which a viral vector infects tissues or cells other than the desired tissue or cell. Mutations that achieve both of these effects, and their use to improve delivery to desired or targeted tissues or cell types are described herein. Further, it has surprisingly been found that one can combine, within a single viral capsid polypeptide, mutations that improve tropism for a desired or targeted tissue or cell type with mutations that reduce tropism for a non-desired tissue or cell type, and thereby further improve targeting efficiency of the vector.

Viruses are typically tropic for certain types of cells and or tissues in the natural host. Changes in this tropism, either increased or decreased, may be determined by selection on a natural host cell or tissue or selection on a non-natural host cell or tissue. Indeed, experiments were performed to select for increased infectivity for the following tissues: blood, brain, heart, kidney, liver, lung, and spleen. Substitution, deletion or insertion mutations that increased the infectivity in these tissues were found. These determinations were also combined with the ability of the mutant plasmids to be packaged into virions.

As detailed below herein, particular mutations have been identified and assessed for effects on viral DNA packaging and viral infectivity of particular tissues. These particular mutations are in the capsid protein. Isolated and purified compositions comprising mutant capsid proteins and nucleic acids encoding them may be used for further viral improvement, for virus preparation and manufacture, and for safety and efficacy studies. Once a mutant protein sequence is identified as beneficial, any nucleic acid codon or codons that specify such protein sequence can be used. Mutations can be combined together in a single viral nucleic acid or a single viral protein sequence for improved properties. The following describes mutations to viral capsid polypeptides that permit viral genome packaging, yet modify viral vector tropism, either positively, negatively, or both when mutations are combined, relative to given tissues or cell types. Also described are methods of using mutated viral capsid polypeptides and viral vectors comprising them to introduce nucleic acids to desired tissue or cell types with improved selectivity for those tissue or cell types. The following provides a description of the various mutations and considerations for their use to generate viral vectors with improved properties.

Generating Variants and Viral Particles

A DNA library of AAV capsid variants was created. Initially each mutant capsid was generated with a single mutation. All possible single amino acid substitutions, insertions and deletions for AAV2 were generated. In a subsequent step, several mutations were combined within the capsid gene. Libraries of AAV2 capsid gene sequence variants were cloned into a plasmid containing the AAV Inverted Terminal Repeat regions (ITRs). The final ITR plasmids contained a cytomegalovirus (CMV) promoter upstream of the Cap gene.

AAV virus libraries were produced from the DNA libraries. The capsid library plasmids, AAV pHelper plasmids, and plasmids containing the AAV2 Rep gene were co-transfected into HEK-293 cells using PEI. Capsids were purified using standard techniques for cell lysis (freeze-thaw or addition of 5 M NaCl), treatment with benzonase to remove unpackaged genomes, and purification and concentration by iodixanol ultra-centrifugation

In vivo packaging ability of viral capsid variants was measured. The number of viruses that were packaged (“virus”) compared to the number of input viral genomes (“plasmid”) were determined. Measuring the frequency of capsid (or other library component) mutants before and after selection reveals which mutations are beneficial and which are deleterious based on the particular selection method.

Evaluating Viral Tropism

To investigate viral tropism, virus libraries were injected into mice intravenously. Blood was collected 1 hour after injection and tissue samples were collected 1-2 weeks after injection. Viral DNA was extracted from bulk biological samples using standard techniques.

The frequencies of capsid variants in biological samples, the virus libraries and the DNA libraries were measured using high-throughput DNA sequencing. Mutant frequencies were normalized by dividing the number of reads matching each mutant by the number of reads matching the AAV2 WT sequence within each sample.

Selection values, indicating enrichment or de-enrichment within a particular sample, are calculated relative to an initial library. Selection values greater than 1 indicate enrichment relative to WT. Selection values less than 1 indicate de-enrichment relative to WT.

For packaging selection, the frequencies in the viral library were compared to those in the DNA library as a reference. Enrichment means that a mutant packaged more efficiently than the WT.

For tissue selection, the frequencies in tissue samples were compared to those in the viral library as a reference. Enrichment means that a mutant was delivered more efficiently than the WT to the specified tissue. De-enrichment means that a mutant was delivered less efficiently that WT to the specified tissue.

Viral Capsid Polypeptide

Viral tropism refers to the cell or tissue type(s) in a host that recruit and support the growth of a virus. Various factors impact the viral tropism, including the expression of cell surface receptors and/or ligands, transcription factor expression, and expression of tropogens (e.g., cell surface glycoproteins). One aspect of the invention provides a viral capsid polypeptide bearing a mutation relative to wild-type adeno-associated virus 2 (AAV2, e.g., SEQ ID NO: 1) that alters tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 1-9. In one embodiment, the tissue is blood, heart, kidney, liver, lung, or spleen.

In one embodiment, the tropism is increased, e.g, the virus comprising the mutated viral capsid polypeptide more efficiently delivers nucleic acid to the target cell type as compared a virus comprising the wildtype viral capsid polypeptide. In one embodiment, tropism is at least 1.1-fold (e.g., 10% greater than reference level, or 110% of the level reference level) more efficient as compared to a wild-type viral capsid polypeptide. In one embodiment, the the delivery of a nucleic acid is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 5-fold, at least 10-fold, or more, more efficient as compared a virus comprising the wildtype viral capsid polypeptide. One of ordinary skill in the art can measure the delivery efficiency of a viral particle comprising any of the viral capsid polypeptides described herein, e.g., using PCR-based assays on an isolated targeted cell or tissue type (e.g., blood, heart, kidney, liver, lung, or spleen) to assess if the nucleic acid is expressed in that targeted cell type. The expression of the nucleic acid delivered by a viral particle comprising either a viral capsid polypeptide as described herein or a wild-type viral capsid polyprotein can be compared to determine the change in expression as a measure of the efficiency of delivery.

In one embodiment, the tropism is decreased, e.g, the virus comprising the mutated viral capsid polypeptide less efficiently delivers nucleic acid to the target cell type as compared a virus comprising the wildtype viral capsid polypeptide. In one embodiment, the tropism is decreased by at least 10%. In other embodiments, the tropism is decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or more as compared to a virus comprising the wildtype viral capsid polypeptide. Methods for measuring tropism are described herein above.

The following describes single amino acid capsid alterations that increase the tropism of a virus (e.g., AAV2) for a given tissue or cell type, e.g., blood, heart, kidney, liver, lung, or spleen. In one embodiment, a single mutation described herein is introduced to the amino acid sequence of wild-type AAV2 capsid protein (e.g., SEQ ID NO: 1) to increase or decrease tissue or cell type tropism or a virus.

SEQ ID NO: 1 is an amino acid sequence encoding wild-tvpe AAV2 capsid protein.

(SEQ ID NO: 1) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYK YLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQE RLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEP DSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMA TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNH LYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGF RPKRLNFKLFNIQVKEVTQNDGITTIANNLTSTVQVFTDSEYQLPYVLGSA HQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNN FTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSR LQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHL NGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITD EEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVY LQGPIWAKIPHIDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTIFSA AKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTV DINGVYSEPRPIGTRYLTRNL

As used herein, “mutation” refers to any change in the amino acid sequence, e.g., a substitution, insertion, or deletion of at least one amino acid. For the mutations described herein, a substitution at a particular position is denoted by the wildtype amino acid followed by the position of the substituted amino acid followed by the identity of the substituted amino acid(s) in a parenthetical, e.g., Q101(A) means glutamine is substituted with alanine at position 101. If multiple amino acids can be substituted at a particular position, the parenthetical lists all possible single substitutions, e.g., P31(IKRT) means that proline at position 31 is substituted with any one of isoleucine, lysine, arginine, or threonine. To denote an insertion, the amino acid position contains a decimal followed by an amino acid(s); the amino acid(s) following the decimal are inserted following the position indicated, e.g, 28.5(AGVY) means any one of alanine, glycine, valine, or tyrosine is inserted immediately after amino acid 28 and immediately before amino acid 29. To denote a deletion of an amino acid, “(-)” follows the amino acid position, e.g., A35(-) means amino acid 35 (alanine) is deleted from the sequence.

The following provides mutations to the AAV2 capsid polypeptide of SEQ ID NO: 1 that provide tropism altered by the indicated degree for cells of the indicated tissues.

TABLE 1 Amino acid alterations conferring more efficient delivery to blood. Fold increase of Amino acid alteration efficiency D4(F), D13(HQ), Q21(FS), W22(F), W23(EGLTVY), 23.5(Y), L25(C), 1.1-fold K26(N), 28.5(AGVY), P29(DEGSTVY), P31(IKRT), 32.5(F), K33(AFLPST), 33.5(IP), 34.5(EY), A35(—), E36(CDGV), 36.5(CW), R37(GV), 37.5(DHNW), H38(K), 38.5(DHQ), G49(W), V65(KT), K77(EG), D80(KW), R81(TY), Q82(AIL), L83(Q), D84(N), S85(FQR), G86(Q), N88(FH), K92(Q), H95(W), A98(Q), E99(T), Q101(A), E102(HIKT), K105(Y), E106(T), 107.5(F), T108(W), F110(QTW), G112(A), N113(Y), R116(AK), A117(C), Q120(DH), R124(AQ), V125(HITW), P128(G), L129(E), 129.5(P), L131(C), V132(D), 132.5(D), E134(N), 134.5(K), P135(-I), V136(AI), K137(E), 138.5(HKP), A139(FHWY), 139.5(IK), 141.5(FG), 144.5(P), P145(FG), E147(T), 147.5(Y), H148(-Q), S149(DFT), 151.5(I), E152(Y), 152.5(AV), S157(H), T159(H), A162(K), Q164(HRSW), Q165(I), P166(HNQ), 177.5(C), A179(P), 179.5(W), V182(L), D184(W), 188.5(CF), G189(CHQSY), 189.5(AEF), Q190(FNVY), 190.5(CE), P191(FH), 191.5(N), P192(NR), 192.5(F), A193(KQ), A194(-NQ), 194.5(EQW), P195(—), T202(WY), T205(CDF), A209(C), N214(D), G217(D), 237.5(N), A248(C), Q263(T), S264(G), Y272(F), R310(K), N312(E), T324(S), 324.5(R), D327(E), T344(EGHY), S346(Q), L380(V), S384(A), Q401(N), S412(A), 417.5(S), V418(C), L433(V), 445.5(D), S446(—), 446.5(DE), R447(-PWY), 447.5(DEHQ), T448(-DEH), 448.5(D), N449(H), T450(FS), 452.5(P), G453(V), T454(APQ), T455(GMS), T456(A), 456.5(A), 457.5(MT), Q464(R), 464.5(Y), S468(M), C482(MSW), Y483(W), R484(ILQV), 485.5(M), Q486(DE), R487(ACDEFGILMNPQSTVWY), 488.5(DE), S489(DE), 490.5(DE), T491(FN), 491.5(DE), A493(FS), 493.5(ENST), 494.5(E), N495(DEW), 495.5(DE), N496(DE), 496.5(DE), N497(AMP), 498.5(ST), 500.5(D), S501(D), 501.5(DE), 502.5(DE), 503.5(DE), G504(DE), 504.5(E), 505.5(E), K507(-EF), P521(I), M523(C), S525(T), 525.5(DE), K527(PQW), D528(Q), E531(DGMN), K532(ADEFGILMNPQSTVY), 532.5(DEMN), F533(DEY), 533.5(CDEGSV), 536.5(V), V539(C), V571(DE), Q575(A), Y576(M), 577.5(E), V579(VV), 579.5(CDEN), S580(ADE), 580.5(DEILMPY), T581(DSWY), 581.5(DEFILMPQV), N582(-ACDEPQ), 582.5(GP), L583(-DEGPT), 583.5(DEIPV), Q584(-DEFLNTY), 584.5(ADEIMVY), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-DEP), 586.5(DEFHINVY), N587(-DE), 587.5(DEGPY), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEFQTY), Q589(DEFMNY), 589.5(DEGINS), A590(DEW), 590.5(DEFY), A591(EFPQSY), 591.5(CDEQWY), T592(FY), 592.5(DEQ), A593(GLMTVW), 593.5(CDEILY), 594.5(CDEP), 595.5(C), N596(S), T597(AS), V600(AILST), L601(L), G603(G), V605(C), D608(N), R609(R), V611(C), P616(M), H623(INY), T624(C), L639(C), H641(S), I646(C), N656(AH), 657.5(Q), T660(H), S662(HWY), 662.5(I), A664(W), F666(M), A667(FR), K688(P), V708(WY), and N734(S) 445.5(D), S446(—), 446.5(DE), R447(-P), 447.5(DEHQ), T448(-DE), 1.5-fold N449(H), T454(P), 464.5(Y), C482(M), R484(ILQV), 485.5(M), Q486(DE), R487(ACDEFGILMNPQSTVWY), 488.5(E), S489(DE), 490.5(DE), T491(N), N495(DEW), 495.5(DE), N496(DE), 496.5(DE), S501(D), 501.5(D), 502.5(DE), 503.5(DE), G504(DE), K507(-E), 525.5(D), E531(GMN), K532(ADEFGILMNPSTVY), 532.5(DEM), F533(DE), 533.5(CDEGSV), V571(DE), 577.5(E), V579(V), 579.5(CDE), S580(DE), 580.5(DEILMY), T581(W), 581.5(DEFILMPQV), N582(-ACDEP), 582.5(GP), L583(-DEPT), 583.5(DEIPV), Q584(-DEFLTY), 584.5(DEIV), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-DE), 586.5(DEFHY), N587(-DE), 587.5(DEP), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEFY), Q589(DEFY), 589.5(DEIN), A590(DEW), 590.5(DE), A591(PY), 591.5(CDEY), T592(FY), 592.5(DE), 593.5(CDE), 594.5(CDE), 595.5(C), N596(S), and A667(FR) 445.5(D), S446(—), 446.5(DE), R447(-P), 447.5(EHQ), T448(D), T454(P), 2-fold 464.5(Y), R484(ILQV), 485.5(M), Q486(DE), R487(ACDEFGILMNPQSTVWY), 488.5(E), S489(DE), 490.5(D), N495(DEW), 495.5(DE), N496(DE), 496.5(DE), S501(D), 501.5(D), 502.5(E), 503.5(DE), G504(DE), K507(—), K532(ADEGILMNPSTV), 532.5(DE), F533(DE), 533.5(CDEGSV), V571(DE), 577.5(E), 579.5(DE), S580(DE), 580.5(DEILMY), T581(W), 581.5(DEFILMPQV), N582(-DEP), 582.5(GP), L583(-DEP), 583.5(DEIPV), Q584(-DEFY), 584.5(DEIV), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-DE), 586.5(DEY), N587(-DE), 587.5(DEP), R588(-ACDEFGHILMNPQSTVWY), 588.5(DE), Q589(DE), 589.5(DEIN), A590(DEW), 590.5(DE), A591(PY), 591.5(DE), T592(FY), 592.5(DE), 593.5(DE), and 594.5(CDE) N496(DE), S501(D), G504(D), K532(DE), 581.5(DEIPV), N582(DE), 5-fold 582.5(G), L583(-DE), 583.5(DEP), Q584(-DE), 584.5(DE), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-D), 586.5(DE), N587(-DE), 587.5(DEP), R588(-ACDEFGHILMNPQSTVY), 588.5(DE), 589.5(DE), A590(DE), 590.5(D), 593.5(E), and 594.5(E) R585(D) 10-fold

TABLE 2 Amino acid alterations conferring more efficient delivery to heart. Fold increase of Amino acid alteration efficiency E17(D), W23(GKRY), 23.5(Y), L25(C), P29(D), P31(INRT), K33(LP), 1.1-fold 33.5(P), A35(E), 35.5(EKPQ), E36(DG), 36.5(PS), R37(K), 37.5(HSW), H38(K), 38.5(D), K39(Q), G49(W), A70(KW), K77(EKQ), R81(Y), Q82(I), S85(FQ), K92(Q), A98(Q), Q101(AN), R103(T), E106(W), T108(W), L114(F), R116(FIKQ), V118(AFV), A121(T), G130(EW), L131(C), E134(DNP), 134.5(KLS), P135(—), 135.5(V), V136(R), 136.5(A), 138.5(H), 140.5(PV), 141.5(GP), V146(G), H148(-Q), 148.5(V), P150(FN), 150.5(Y), V151(IN), 151.5(EI), E152(F), 152.5(VW), T159(K), K161(F), A162(KN), Q164(AEFW), N172(Y), 179.5(W), D180(NW), V182(I), D184(W), Q186(F), 188.5(T), G189(DEQY), 189.5(DEFIKV), Q190(-F), 190.5(EIQT), P191(H), 191.5(N), P192(G), 192.5(FK), A193(—), A194(-ENT), 194.5(EW), P195(—), T205(F), S243(C), I260(L), Q263(A), S264(G), S267(S), R310(K), D327(E), T344(EP), S346(Q), S356(N), Q359(D), S384(S), V387(M), R389(S), T414(V), V418(C), S422(G), L433(A), I438(N), Y444(F), 448.5(S), N449(A), T450(M), S452(M), T454(LQ), 455.5(A), T456(AV), Q457(DLV), S458(AP), 458.5(Q), R459(FH), Q464(AI), A467(CGNPQ), S468(ADG), D469(AENQST), I470(LMV), R471(CKM), C482(M), Q486(N), R487(ACGIMQSTV), S489(AD), 490.5(N), T491(FLW), 492.5(M), A493(M), N496(DHSY), N497(PV), S498(FM), Y500(M), T506(S), V517(I), S525(AG), H526(CNST), E531(D), K532(ADEFGILMNQRST), I541(C), K544(CGTVY), Q545(E), K549(Q), N551(Y), V552(Q), K556(DNY), D561(Q), R566(ASV), T567(S), S578(TV), V579(V), S580(A), T581(WY), 581.5(IV), N582(DE), L583(DE), 584.5(EIV), R585(- ADFGHILMNQSTVWY), G586(DE), N587(DE), 587.5(D), R588(ACDEFHILMNQSTVY), 588.5(DE), Q589(M), A590(P), A591(ETY), 591.5(CDE), T592(F), 594.5(D), T597(ANSW), Q598(L), V600(AS), W606(F), V611(C), T624(C), H641(QS), L647(F), N656(A), P657(P), S658(P), T659(A), S662(FQ), A664(L), A667(N), Q687(K), V708(WY), N709(A), and N734(P) 37.5(H), S264(G), D327(E), R389(S), D469(AQS), I470(LM), R471(K), 1.5-fold R487(A), S489(D), 490.5(N), T491(W), N496(D), S498(M), K532(ADEL), D561(Q), N582(DE), L583(E), 584.5(V), R585(-AGHIMNQSTVWY), N587(DE), R588(ADEFILMNQSTVY), 588.5(DE), Q589(M), 591.5(D), T597(W), and V600(A) R471(K), K532(D), N582(D), L583(E), R585(Q), N587(DE), R588(MQTV), 2-fold and 588.5(E)

TABLE 3 Amino acid alterations conferring more efficient delivery to kidney. Fold increase of Amino acid alteration efficiency D13(Q), E17(D), Q21(F), W23(G), L25(C), 28.5(Y), P29(D), P31(KRST), 1.1-fold K33(ART), 34.5(AY), 35.5(E), E36(DGV), 36.5(S), R37(GK), 37.5(DHSW), H38(KN), 38.5(Q), E63(S), V65(K), A70(K), K77(KQ), A78(E), D80(W), R81(HTY), Q82(I), S85(FGQ), N88(V), K92(Q), E99(A), Q101(N), E102(T), R103(T), E106(W), F110(K), G112(AS), R116(IKQ), V118(AF), Q120(FH), R124(E), L126(DH), E127(L), L129(A), 129.5(P), G130(E), L131(Q), E134(P), 134.5(KPRSWY), P135(—), 135.5(V), V136(AR), 137.5(K), 138.5(PW), 140.5(PV), 141.5(A), 145.5(FY), V146(P), 146.5(E), 147.5(Y), H148(-EQ), S149(D), P150(F), 150.5(Y), V151(IN), 152.5(V), P153(P), A162(T), Q164(EL), Q165(I), D180(NW), S181(E), Q186(WY), L188(F), 188.5(Q), G189(DEQ), 189.5(AKN), Q190(FY), 190.5(EQT), P191(KPP), 191.5(F), P192(V), 192.5(EIK), A193(—), 194.5(DE), T200(Y), N201(HY), T205(D), G217(D), Q263(A), S264(G), R310(K), D327(E), T344(AHP), V387(M), R389(S), T410(Q), A425(S), 446.5(D), 447.5(HQ), N449(ACGS), T456(AG), 456.5(G), R459(HK), Q464(A), A467(GNP), S468(AG), D469(AEQST), I470(LMV), R471(K), C482(M), Q486(D), R487(P), S489(E), 490.5(N), T491(F), A493(FI), N495(E), 495.5(DE), N496(EHS), 496.5(D), N497(P), S498(M), Y500(M), S501(D), 501.5(D), G504(D), N518(S), H526(CS), K532(DER), 532.5(D), F533(Y), K544(ILTV), T550(G), K556(N), D561(Q), R566(AV), S578(TV), V579(V), S580(A), 580.5(IMY), 581.5(EFILMPQV), N582(-ACDEP), 582.5(GP), L583(-DEIP), 583.5(DEIPV), Q584(-EFY), 584.5(DEILV), R585(-DEINPQVW), 585.5(D), G586(—), 586.5(DEIY), N587(-D), 587.5(DEP), R588(-ACDEGHLNPQTV), 588.5(DE), Q589(MP), 589.5(DE), A590(DEPV), 590.5(DE), A591(PST), 591.5(C), A593(M), 593.5(CE), T597(ASW), Q598(L), V600(A), V611(C), H623(IY), H627(F), P630(M), L639(C), H641(NQS), S658(AP), T659(V), F666(M), and V708(WY) R389(S), D469(AQ), I470(L), R471(K), 490.5(N), 495.5(DE), N496(EHS), 1.5-fold S498(M), S501(D), K532(D), 580.5(I), 581.5(EILMPV), N582(-D), 582.5(GP), L583(DE), 583.5(DEIPV), Q584(-EFY), 584.5(IL), R585(-IP), G586(—), N587(—), 587.5(P), R588(-CDGV), 588.5(D), 589.5(DE), A591(P), 591.5(C), 593.5(C), T597(W), and V600(A) R389(S), R471(K), K532(D), 581.5(IM), L583(DE), 583.5(D), 584.5(L), 2-fold G586(—), N587(—), 587.5(P), R588(—), 591.5(C), and 593.5(C)

TABLE 4 Amino acid alterations conferring more efficient delivery to liver. Fold increase of Amino acid alteration efficiency E17(D), P31(Q), K33(HV), 35.5(KW), E36(D), 36.5(EY), R37(QT), 37.5(H), 1.1-fold H38(K), A70(EG), K77(Q), A78(DE), D80(KW), R81(HY), Q82(A), L83(I), D84(N), S85(FGQ), N88(F), L91(Y), A98(Q), E99(AL), Q101(NT), E102(KT), R103(QW), K105(D), E106(A), 107.5(F), F110(QT), V118(CFV), Q120(DN), R124(Q), V125(IT), L126(T), E127(S), L129(HP), 129.5(P), 133.5(T), E134(HRV), 134.5(S), P135(L), 139.5(IQ), P140(I), G141(P), K142(V), 145.5(A), H148(FP), 148.5(D), 149.5(D), 150.5(H), 151.5(K), E152(S), 152.5(A), P153(P), S157(Y), T159(KN), 163.5(Q), Q164(AF), 177.5(F), D178(C), A179(Q), G189(DFQS), 189.5(HISTVY), Q190(F), 190.5(EKLN), P191(GT), 191.5(DN), 192.5(A), A193(—), A194(N), 194.5(W), S196(A), T205(N), A212(E), A248(C), I260(M), Q263(A), S264(G), Y275(W), Q325(T), D327(E), T329(H), T331(Q), T344(AHPY), S356(GN), M371(H), V387(M), R389(S), T414(Y), S423(S), L433(AC), I438(CS), Y444(F), N449(S), T450(IV), S452(A), 455.5(A), T456(V), Q457(T), S458(AP), R459(HKT), L460(CN), A467(CGPQ), S468(G), D469(AENQST), I470(LMV), R471(CKM), C482(IM), Y483(M), Q486(N), 490.5(N), T491(DE), A493(MP), N496(HS), S498(AFLM), Y500(M), T503(V), T506(ACV), K507(S), V517(I), S525(G), H526(ACNST), K532(HINQWY), F533(Y), K544(C), 545.5(D), T550(E), I554(L), K556(ENY), M558(F), D561(Q), E563(D), R566(ACGNSTV), T567(S), S578(DEIT), V579(V), S580(A), T581(DM), L583(I), A590(IP), A591(DEQS), T592(S), A593(DET), N596(C), T597(ACDHLNQW), Q598(ILMV), V600(AST), W606(F), Q607(M), V611(C), H623(NQ), T624(A), L639(C), H641(STVW), N656(H), 657.5(A), S658(W), 658.5(T), S662(F), A663(D), A664(PS), F666(HM), Q687(K), V708(FW), N709(A), and N734(P) L91(Y), V118(V), G189(F), S264(G), Y275(W), V387(M), R389(S), 1.5-fold R459(H), L460(N), A467(CGPQ), D469(AEQST), I470(LM), R471(K), C482(I), Q486(N), N496(H), S498(M), H526(CNS), K532(N), K544(C), T550(E), K556(Y), R566(AGTV), A590(P), A591(E), A593(DE), T597(HNQ), V611(C), V708(W), and N709(A) R389(S), A467(G), D469(AEQS), I470(L), R471(K), Q486(N), N496(H), 2-fold S498(M), H526(S), T550(E), A593(DE), and T597(N)

TABLE 5 Amino acid alterations conferring more efficient delivery to lung. Fold increase of Amino acid alteration efficiency E17(D), W23(T), L25(C), K33(T), 35.5(DE), E36(DV), 36.5(AP), 1.1-fold 37.5(DSW), H38(N), 38.5(DQ), K39(P), 42.5(Y), E63(DR), A70(K), K77(EQR), R81(L), L83(I), D84(N), S85(FQ), K92(Q), A98(FQ), E99(V), Q101(N), E102(T), K105(F), F110(K), R116(FIQ), V118(V), E127(GL), L129(A), 129.5(P), 134.5(HKS), 135.5(PV), V136(P), 137.5(K), 140.5(APT), 141.5(AP), 145.5(FY), V146(HI), H148(-EQW), S149(W), 149.5(D), P150(I), V151(N), 151.5(NS), E152(S), 152.5(V), P153(P), S157(W), T159(EIQ), K161(F), A162(W), Q164(DEHLW), S181(E), D184(W), Q186(Y), L188(F), G189(DEQ), 189.5(DEK), 190.5(EINY), P191(GP), 192.5(P), A193(Q), A194(N), 194.5(D), T205(D), N214(D), Q263(AN), S264(G), S267(S), R310(K), T324(S), D327(E), T329(H), T344(AP), S356(N), V387(M), R389(S), T410(DQ), T414(R), V418(C), Y444(F), N449(Q), T450(I), S452(M), T454(IQ), T455(G), S458(AP), 458.5(Q), R459(F), Q461(G), A467(CGNPQ), S468(G), D469(AENQST), I470(LMV), R471(K), 490.5(N), T491(QW), N497(P), N518(M), P521(V), F533(Y), V539(C), K544(C), Q545(N), T550(A), K556(N), D561(Q), S578(T), S580(A), Q589(M), A590(P), A593(T), T597(AS), V600(A), W606(F), V611(C), H623(NY), L639(C), H641(NQS), 1646(C), N656(A), P657(P), S658(AP), 658.5(Q), T659(AHV), S662(FQ), A667(N), S679(C), V708(W), and N709(A) S264(G), A467(G), D469(AQT), I470(M), R471(K), 490.5(N), and V600(A) 1.5-fold

TABLE 6 Amino acid alterations conferring more efficient delivery to spleen. Fold increase of Amino acid alteration efficiency E12(C), E17(D), Q21(DF), W23(G), 23.5(Y), P29(Y), P31(IT), 33.5(P), 1.1-fold E36(D), 36.5(C), 37.5(HW), H38(K), 38.5(Q), E63(PS), V65(K), K77(E), D80(GKW), R81(GLTY), Q82(I), L83(I), D84(EN), S85(FQR), N88(F), H95(W), A98(Q), E99(AT), E102(IT), K105(F), E106(W), 107.5(F), T108(W), F110(KQW), G112(A), N113(Y), R116(F), Q120(HK), R124(A), V125(T), L129(E), 129.5(P), G130(ALW), L131(C), 132.5(D), 133.5(I), E134(N), 134.5(KRS), P135(—), 135.5(PVY), V136(—), 136.5(I), 138.5(GHP), A139(FHNVW), 139.5(F), P140(E), 140.5(W), G141(E), 141.5(FG), K142(N), 142.5(1), 144.5(P), P145(F), V146(P), 147.5(LQVY), H148(QY), 148.5(W), S149(D), 149.5(P), P150(Y), 150.5(Q), 1515(IP), E152(WY), 152.5(A), P153(P), A162(K), Q164(EPRS), P166(Q), A179(P), D184(W), 188.5(D), G189(DEQVY), 189.5(AHY), Q190(F), 190.5(ELQY), 191.5(D), P192(NRY), 192.5(F), A193(K), A194(N), 194.5(EW), P195(—), N201(Y), T205(CDHN), A209(C), N214(D), E216(D), Q263(N), S264(A), Y272(F), N312(K), D327(E), 327.5(G), T344(AEHPY), F370(F), M371(H), V372(I), Q401(N), L445(I), S458(A), R459(K), S468(G), D469(Q), T491(LQ), Y500(W), F533(Y), 1541(C), M558(F), V579(V), S580(A), Q589(MN), A590(P), A591(S), A593(MT), T597(AMY), V600(AIT), V605(C), W606(F), D608(N), V611(C), H623(Y), T624(C), L639(C), H641(NQS), 658.5(T), T659(N), S662(H), 662.5(1), A664(Q), A667(F), T716(A), and N734(A) Q589(M), and V600(A) 1.5-fold

TABLE 7 Amino acid alterations that reduce delivery to liver. Fold increase of Amino acid alteration efficiency 4.5(HNV), 21.5(F), 23.5(E), 42.5(T), 45.5(M), K51(Y), Y52(D), P55(Y), 0.1-fold G58(C), 82.5(D), 98.5(C), 144.5(N), P166(K), G174(H), D180(M), 180.5(M), 183.5(M), 184.5(M), G217(F), Q259(P), Q263(—), S264(—), 265.5(D), S267(E), 267.5(E), N268(E), 268.5(E), 269.5(D), N270(HY), R307(Y), 324.5(K), 379.5(DE), L380(E), N381(DMQ), 382.5(D), 383.5(E), 386.5(D), V387(E), G406(E), 412.5(Q), 431.5(Y), R432(—), D439(K), 444.5(CM), 445.5(ACDEGHILMNQS), S446(—), 446.5(ACEFGHIMNPQSTVWY), R447(-IPWY), 447.5(DEFGHIMNPQVW), T448(-DEGHILMNPQVWY), 448.5(DENP), N449(P), 449.5(E), G453(K), 453.5(Y), T454(F), 454.5(Y), T456(H), L460(WY), 460.5(W), S463(QV), 463.5(F), 469.5(E), R471(EG), D472(MT), S474(I), 475.5(SW), 476.5(L), 478.5(L), P479(T), 479.5(D), G480(Y), C482(W), 483.5(QV), R484(EFGHILMSVY), 484.5(N), 485.5(IKM), Q486(AFIMVWY), 486.5(FV), R487(DEFPY), 487.5(FW), V488(EH), 488.5(DEILMPQSWY), S489(-EFHKLMPQTY), 489.5(DGMPQRT), K490(-P), 490.5(HPQ), T491(P), 491.5(PW), 492.5(H), A493(H), 493.5(H), D494(FR), 494.5(AFGHILMNPVY), N495(-ADEFGHILPQSTVWY), 495.5(ACDFGILMNPSTV), N496(-EGL), 496.5(ADEFGHILNPSVY), N497(—), 497.5(ADEFGIPQSY), S498(-CEIPR), 498.5(DEGNPSVY), E499(CI), 499.5(ACDEGILMNPQSTVWY), Y500(-ACEGIKNP), 500.5(ADEFGHILMNPQSTVWY), S501(-IL), 501.5(ACDEFGHILMNQRSTVWY), W502(I), 502.5(ADEFGMNY), T503(E), 503.5(DEFGILMNQSW), G504(-DEHILMNPQSVWY), 504.5(ACDFHILMNPSTVWY), A505(DEILMNPY), 505.5(ACDEFGILMQTV), T506(M), 506.5(ACISVW), K507(-EF), 510.5(K), N511(EY), 523.5(A), 524.5(CGHST), 525.5(ACDFGILNPQSTVWY), H526(DKLP), 526.5(ACDEFGHMPQSVWY), K527(-DW), 527.5(ADEGHILMPQSVWY), D528(FPSW), 528.5(DPT), D529(ES), 529.5(DGLMNV), E530(NPW), 530.5(AEFIMNQTVWY), 531.5(ADFIKLNPQRSTVY), K532(—), 532.5(ACDFGHIKLMPQSTVWY), F533(D), 533.5(ACDFGHILNPQSTVWY), 534.5(F), P535(-F), Q536(-PW), 536.5(CITV), V571(DH), T573(IW), 573.5(T), 575.5(WY), Y576(V), 576.5(W), G577(D), 577.5(DEHIQS), 578.5(CDEILTVY), V579(DW), 579.5(DEINQTVY), S580(-DEQY), 580.5(DMNV), T581(—), 581.5(CDGILMPQY), N582(-FHP), 582.5(DFGHPTVW), 583.5(DEGMNSTVY), Q584(-DEFGHLNPVWY), 584.5(CEILP), R585(D), 585.5(DGI), G586(—), 586.5(ADFILMNQSV), N587(-H), 587.5(ADHMPQTW), R588(-GP), Q589(-D), 589.5(CDEHKNTV), A590(DEFKLMN), 590.5(DEKMQTWY), A591(—), 59E5(GHI), 592.5(ACEGILMTWY), A593(-P), 593.5(AFLMNPQSTVWY), 594.5(ACFGILMPTVWY), 595.5(ACFGILMS), 596.5(EY), 597.5(F), 600.5(F), 606.5(P), D608(W), D610(Y), Q614(T), 617.5(I), S679(F), 705.5(Q), K706(ES), V710(M) 4.5(FHNV), G5(C), L11(C), E17(F), G18(D), I19(N), 21.5(F), 23.5(DEN), 0.2-fold 24.5(H), L25(GH), 25.5(CKY), 28.5(T), P30(DY), 30.5(DKLRY), P32(E), 37.5(M), 42.5(T), 45.5(M), K51(Y), Y52(D), P55(Y), 55.5(K), G58(CE), G62(Q), 79.5(C), 82.5(D), G86(C), 87.5(Q), 90.5(E), Y93(N), 98.5(CE), 103.5(V), K105(M), 116.5(E), 123.5(FQ), 125.5(ES), 132.5(W), K143(Y), 144.5(N), P145(-E), 148.5(M), S156(T), 157.5(N), G163(K), Q164(M), P166(CIK),N172(W), 173.5(NQ), G174(H), D180(M), 180.5(M), 183.5(M), 184.5(M), 189.5(M), G217(F), T251(V), Q259(P), S261(ANT), Q263(-W), S264(-EVW), 265.5(D), 266.5(E), S267(EN), 267.5(E), N268(EQ), 268.5(E), 269.5(D), N270(HQY), H271(EIV), F273(HW), R307(Y), 324.5(K), Q349(AS), F365(W), L378(V), 379.5(DE), L380(E), 380.5(E), N381(ACDGMQS), N382(S), 382.5(D), 383.5(E), 386.5(D), V387(EW), 387.5(P), G406(DEQ), 406.5(Q), N408(G), 412.5(Q), 417.5(M), H426(N), Q428(EH), 431.5(Y), R432(—), D439(K), 444.5(CLM), 445.5(ACDEGHILMNQST), S446(-ELNV), 446.5(ACDEFGHILMNPQSTVWY), R447(-DEFIPWY), 447.5(ACDEFGHILMNPQVWY), T448(-DEFGHILMNPQVWY), 448.5(DEFHNPY), N449(-P), 449.5(E), T450(—), 450.5(E), 451.5(N), G453(FK), 453.5(INY), T454(F), 454.5(DY), T456(H), 456.5(E), R459(P), 459.5(P), L460(WY), 460.5(W), S463(KQV), 463.5(F), 464.5(SY), 466.5(E), 469.5(E), R471(EGQ), D472(MT), S474(I), R475(H), 475.5(SW), 476.5(L), W477(F), 477.5(L), 478.5(L), P479(T), 479.5(D), G480(Y), 481.5(G), C482(W), 483.5(QV), R484(EFGHILMQSTVY), 484.5(N), 485.5(EIKM), Q486(ACEFILMPVWY), 486.5(FV), R487(DEFKLMPSTY), 487.5(FW), V488(EGHMNY), 488.5(ACDEGILMPQSWY), S489(-EFHIKLMNPQTVY), 489.5(ADGLMPQRST), K490(-DP), 490.5(DHPQ), T491(P), 491.5(AEFHLPTVWY), S492(—), 492.5(EGHQ), A493(H), 493.5(EHMNQ), D494(FR), 494.5(AFGHILMNPVY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMNPSTVWY), N496(-EGLMQ), 496.5(ADEFGHILNPSTVWY), N497(-CHW), 497.5(ADEFGIMNPQSTY), S498(-CDEINPRW), 498.5(ADEFGILMNPQSVWY), E499(CIPS), 499.5(ACDEGILMNPQRSTVWY), Y500(-ACEGHIKLNPTV), 500.5(ADEFGHILMNPQSTVWY), S501(-DILP), 501.5(ACDEFGHILMNPQRSTVWY), W502(-CEFILSV), 502.5(ADEFGILMNPSTVWY), T503(AEFGMQ), 503.5(ADEFGILMNPQSTVW), G504(-CDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(DEFHILMNPQTVWY), 505.5(ACDEFGILMPQTVWY), T506(MP), 506.5(ACIPSTVW), K507(-EFY), H509(F), 509.5(K), 510.5(K), N511(ACDELWY), 512.5(S), R513(H), D514(LV), S515(1), 523.5(A), 524.5(CGHST), S525(M), 525.5(ACDFGILMNPQSTVWY), H526(DKLP), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFHPWY), 527.5(ACDEFGHILMPQSVWY), D528(EFMPSVW), 528.5(DEPT), D529(ES), 529.5(DEGLMNPV), E530(NPVW), 530.5(AEFGHIKLMNQRSTVWY), E531(CFIV), 531.5(ADFGIKLMNPQRSTVY), K532(—), 532.5(ACDEFGHIKLMPQSTVWY), F533(ADEGQSV), 533.5(ACDEFGHILNPQSTVWY), F534(I), 534.5(F), P535(-F), Q536(-DFPW), 536.5(ACITV), S537(—), 537.5(T), G538(—), V539(H), 542.5(Q), 544.5(F), S547(—), 548.5(H), 550.5(EKM), 551.5(IMQ), E555(HN), V571(DEFHLMQY), T573(CHIW), 573.5(T), E574(LM), 575.5(WY), Y576(CGHV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMQSV), 578.5(ACDEFILSTVY), V579(DW), 579.5(CDEFGILMNQTVY), S580(-DEFILMNQY), 580.5(ADEFGLMNTV), T581(—), 581.5(ACDEGILMNPQTVY), N582(-AFGHMPWY), 582.5(DFGHPTVW), L583(-DGP), 583.5(DEGMNPSTVY), Q584(-ADEFGHILMNPTVWY), 584.5(CEILP), R585(-DPV), 585.5(DEGILMVW), G586(—), 586.5(ADEFGILMNQSTVY), N587(-H), 587.5(ADEHILMNPQSTVW), R588(-GPWY), 588.5(GHLMP), Q589(-DENTVY), 589.5(ACDEFHKNTVW), A590(DEFHKLMNQY), 590.5(CDEFGIKMQSTWY), A591(—), 591.5(CFGHILMPST), T592(—), 592.5(ACDEFGILMNQSTVWY), A593(-P), 593.5(ACFGILMNPQSTVWY), D594(L), 594.5(ACEFGHILMPQSTVWY), 595.5(ACFGILMNQSVW), 596.5(EY), 597.5(F), G599(W), 600.5(F), 606.5(P), D608(W), D610(Y), 610.5(F), Q614(T), 617.5(1), M634(C), 662.5(W), S679(FIY), E681(D), 704.5(AI), 705.5(Q), K706(EST), 709.5(Q), V710(M), 710.5(D) 4.5(FHNV), G5(-C), 5.5(K), W10(CEKQRS), 10.5(HI), L11(C), 11.5(K), 0.3-fold E12(I), D13(IY), E17(F), G18(D), I19(NQ), 21.5(FY), W22(V), 22.5(IM), W23(D), 23.5(ADEFHLNQT), 24.5(DEHIN), L25(GHN), 25.5(CIKLQVY), K26(Y), 26.5(N), 28.5(EILNST), P29(W), P30(DY), 30.5(DKLRY), P32(EGS), 37.5(M), S42(—), 42.5(T), 43.5(V), 45.5(M), K51(CY), Y52(D), P55(Y), 55.5(KY), G58(CE), D60(C), K61(—), G62(Q), L73(K), 78.5(D), 79.5(C), 81.5(W), 82.5(D), 83.5(Y), 84.5(Q), G86(C), 87.5(EQ), P89(H), 90.5(E), Y93(N), 93.5(Y), A96(QV), 98.5(CE), 103.5(V), K105(M), 105.5(C), 111.5(A), 112.5(CQ), N113(DW), 113.5(N), G115(-FP), 116.5(EQ), 117.5(FQ), V118(DR), 118.5(AC), F119(Q), 120.5(GR), 122.5(Q), 123.5(AFQT), R124(—), 124.5(CHK), 125.5(AEFS), L126(—), P128(F), 129.5(F), 130.5(K), 132.5(W), A139(D), P140(D), K142(D), 142.5(E), K143(VY), 143.5(EPIY), R144(P), 144.5(AN), P145(-E), 145.5(EMT), 148.5(M), 150.5(M), S156(T), 157.5(N), G163(K), Q164(M), 164.5(KP), P166(-CIK), 166.5(T), N172(DW), 173.5(INQ), G174(HTY), 174.5(K), D180(M), 180.5(M), 183.5(M), 184.5(M), G189(M), 189.5(M), Q190(M), P192(M), G217(F), S225(R), H229(D), 237.5(Q), T251(V), Q259(PS), S261(ANT), Q263(-DFW), S264(-DEVW), 265.5(ADS), A266(S), 266.5(AE), S267(ENP), 267.5(E), N268(EQ), 268.5(E), 269.5(D), N270(HQY), H271(EINV), F273(HW), R307(Y), P308(—), 324.5(K), Q325(K), A333(N), Q349(AS), F365(W), A367(P), L378(V), T379(G), 379.5(DE), L380(EMQ), 380.5(E), N381(ACDGMQS), N382(CEST), 382.5(D), 383.5(E), 384.5(D), A386(PT), 386.5(D), V387(EW), 387.5(P), S390(DT), 395.5(W), P399(Q), G406(DEQ), 406.5(Q), N408(DGM), F409(H), 412.5(Q), 416.5(E), 417.5(KM), H426(N), Q428(EH), S429(GP), L430(V), 431.5(Y), R432(-I), D439(KNY), 444.5(CLM), L445(FY), 445.5(ACDEGHILMNQST), S446(-ELNV), 446.5(ACDEFGHILMNPQSTVWY), R447(-DEFILMPVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQVWY), 448.5(DEFGHINPY), N449(-DEHP), 449.5(ELP), T450(—), 450.5(E), 451.5(N), G453(AFKLQ), 453.5(INY), T454(FIN), 454.5(DFY), 455.5(H), T456(DEHN), 456.5(EH), S458(DEW), 458.5(HLMN), R459(-P), 459.5(P), L460(WY), 460.5(W), F462(Y), S463(GKQV), 463.5(F), 464.5(SY), 466.5(E), S468(CD), 469.5(E), R471(DEGQV), D472(LMTWY), S474(I), R475(H), 475.5(SW), 476.5(L), W477(F), 477.5(L), 478.5(L), P479(GT), 479.5(D), G480(Y), 48E5(GHY), C482(W), 483.5(QV), R484(EFGHILMQSTVY), 484.5(N), Q485(AG), 485.5(EIKM), Q486(ACEFILMPVWY), 486.5(FV), R487(ACDEFGKLMNPQSTVY), 487.5(FW), V488(EFGHMNQY), 488.5(ACDEFGILMPQSWY), S489(-EFHIKLMNPQTVWY), 489.5(ADEGLMPQRSTV), K490(-DGP), 490.5(CDHMPQIY), T491(-P), 491.5(AEFGHILPQTVWY), S492(—), 492.5(EGHLQ), A493(-H), 493.5(DEHIKMNPQ), D494(FR), 494.5(AFGHILMNPQTVY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMNPSTVWY), N496(-EGLMPQW), 496.5(ADEFGHILMNPSTVWY), N497(-CGHLWY), 497.5(ADEFGILMNPQSTY), S498(-CDEINPRW), 498.5(ADEFGHILMNPQSVWY), E499(CIPS), 499.5(ACDEGILMNPQRSTVWY), Y500(-ACEGHIKLNPSTV), 500.5(ADEFGHILMNPQSTVWY), S501(-DILPVW), 501.5(ACDEFGHILMNPQRSTVWY), W502(-ACDEFGILMPQSV), 502.5(ADEFGILMNPQSTVWY), T503(-ADEFGHLMNQW), 503.5(ADEFGILMNPQSTVW), G504(-CDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFHILMNPQTVWY), 505.5(ACDEFGILMPQSTVWY), T506(FLMP), 506.5(ACIPSTVW), K507(-EFY), H509(FK), 509.5(K), 510.5(K), N511(ACDEGLMSWY), 512.5(S), R513(H), D514(CFILMV), S515(I), N518(V), P521(M), 523.5(A), A524(G), 524.5(ACGHST), S525(M), 525.5(ACDEFGILMNPQSTVWY), H526(DIKLMPV), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFHPSWY), 527.5(ACDEFGHILMPQSTVWY), D528(EFMPSVW), 528.5(DEPT), D529(ES), 529.5(DEGLMNPV), E530(NPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(CFILV), 531.5(ADFGIKLMNPQRSTVY), K532(-CP), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ADEGIQSV), 533.5(ACDEFGHILNPQSTVWY), F534(ILVY), 534.5(F), P535(-F), Q536(-ADFGHPWY), 536.5(ACILTV), S537(—), 537.5(T), G538(—), V539(H), 542.5(Q), 543.5(P), K544(—), 544.5(FY), 545.5(W), 546.5(Q), S547(—), 547.5(NSY), E548(F), 548.5(CH), 549.5(D), 550.5(EKMQ), N551(P), 551.5(AEIMQ), 552.5(V), E555(HN), D561(I), 565.5(Y), R566(D), 567.5(E), P570(Q), 570.5(M), V571(ADEFHLMQY), T573(ACGHIWY), 573.5(T), E574(CFLM), 575.5(WY), Y576(ACGHIMV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMQSTV), S578(F), 578.5(ACDEFILNSTVY), V579(DW), 579.5(ACDEFGILMNQTVWY), S580(-DEFILMNQY), 580.5(ACDEFGLMNTV), T581(-P), 581.5(ACDEGILMNPQTVY), N582(-ADFGHKMPSWY), 582.5(DFGHNPSTVW), L583(-DGPW), 583.5(DEGMNPSTVY), Q584(-ADEFGHILMNPTVWY), 584.5(CEFGHILPV), R585(-CDILNPVY), 585.5(DEFGILMNPQSTVW), G586(—), 586.5(ADEFGHILMNQSTVY), N587(-EFHY), 587.5(ADEHILMNPQSTVW), R588(-ADGPSWY), 588.5(DFGHLMP), Q589(-DEINRTVY), 589.5(ACDEFHIKMNPTVWY), A590(DEFHKLMNQY), 590.5(ACDEFGIKLMQSTVWY), A591(-P), 591.5(ACFGHILMNPQSTV), T592(-M), 592.5(ACDEFGILMNQSTVWY), A593(-P), 593.5(ACEFGILMNPQSTVWY), D594(LT), 594.5(ACDEFGHILMNPQSTVWY), V595(WY), 595.5(ACDFGHILMNQSTVW), N596(EPQ), 596.5(DEY), T597(P), 597.5(F), Q598(G), G599(W), 600.5(F), L601(W), P602(C), 606.5(P), D608(W), D610(Y), 610.5(F), Q614(T), I617(K), 617.5(1), H629(W), M634(C), 640.5(N), 657.5(C), 658.5(C), 659.5(C), T660(H), F661(I), 661.5(C), 662.5(W), 663.5(CW), 665.5(RW), F666(C), 667.5(Y), S679(FIY), E681(D), 699.5(E), 703.5(AEL), 704.5(AILN), 705.5(QY), K706(EST), S707(L), 707.5(Y), V708(N), 708.5(1), N709(P), 709.5(PQ), V710(M), 710.5(D), D711(G), T716(R), S721(H), P723(D), R724(F) 1.5(M), A3(CD), 3.5(1), 4.5(AFHNVY), G5(-C), 5.5(FKM), 6.5(N), 0.5-fold W10(ACEGKPQRSTY), 10.5(HI), L11(AC), 11.5(K), E12(FI), D13(EIKWY), T14(CDQ), L15(HT), E17(CFVY), G18(D), I19(EGHNQT), R20(Q), 21.5(FWY), W22(IV), 22.5(IMWY), W23(DK), 23.5(ADEFGHLNQSTWY), 24.5(CDEHILN), L25(EFGHNQR), 25.5(CEIKLNQRSTVWY), K26(Y), 26.5(NQ), P27(T), 28.5(AEFILNPQSTW), P29(DEQRSTW), P30(DFHQRSY), 30.5(DEKLNQRTY), P31(H), 31.5(AFV), P32(EGIRS), 32.5(DEKNP), K33(Q), 33.5(CKNVWY), 34.5(FQ), A35(HNQY), E36(H), 36.5(IN), R37(DN), 37.5(M), H38(W), 38.5(G), K39(F), 39.5(DY), D40(-ACNY), 40.5(Y), D41(H), 41.5(NW), S42(-LVW), 42.5(DT), R43(AT), 43.5(PV), 45.5(M), Y50(HW), K51(ACLMVY), Y52(D), P55(Y), 55.5(EKY), 56.5(K), G58(CE), 58.5(F), L59(CGK), 59.5(H), D60(CY), K61(-IS), 61.5(G), G62(Q), A68(T), L73(HK), E74(TW), 77.5(K), A78(FY), 78.5(ADIQ), Y79(C), 79.5(CK), 80.5(F), 81.5(HW), 82.5(DK), 83.5(HKY), 84.5(NQ), 85.5(KS), G86(-C), 86.5(Q), 87.5(DEFQ), P89(CH), 89.5(GL), 90.5(EMY), L91(Q), 92.5(RY), Y93(GN), 93.5(Y), 94.5(Y), A96(QV), A98(C), 98.5(CE), F100(K), 101.5(E), E102(FQ), 103.5(SV), L104(N), 104.5(EKN), K105(M), 105.5(CRY), E106(CF), 106.5(HNY), D107(CKY), 107.5(N), T108(—), 108.5(QY), S109(N), 109.5(Y), 111.5(AW), 112.5(CQ), N113(DIKLRW), 113.5(FN), G115(-DFMPV), 115.5(DHLVW), R116(EP), 116.5(EHIKLPQTWY), Al 17(EKR), 117.5(EFKLPQW), V118(-DKPR), 118.5(ACEGP), F119(CQS), 119.5(AHKLNQRSVY), 120.5(GKLPR), A121(E), 121.5(FG), K122(FNW), 122.5(HIQW), K123(CDHT), 123.5(AFKNQT), R124(-IP), 124.5(CHKPRS), V125(-DPQ), 125.5(ACEFGQRSW), L126(-R), 126.5(CER), E127(—), 127.5(LNSY), P128(CFI), 128.5(AHKTW), L129(DKR), 129.5(F), 130.5(CKSY), 131.5(DEHP), V132(C), 132.5(IW), 135.5(W), 136.5(EM), K137(CDY), 137.5(DLNQSY), 138.5(DNQ), A139(DF), 139.5(D), P140(DE), 140.5(CDEKN), G141(EFKLMQWY), 141.5(DEY), K142(DEGPY), 142.5(DEKPSY), K143(EGVY), 143.5(ELPQSTY), R144(HPSTVW), 144.5(ACFHNSTY), P145(-DENSVY), 145.5(EGMPQT), V146(DE), 146.5(R), E147(FGHKLQY), 147.5(K), 148.5(MR), S149(MPQ), 149.5(CMW), P150(M), 150.5(EM), V151(—), 152.5(HK), S156(AT), 157.5(N), G160(Y), 160.5(N), K161(Y), 161.5(F), G163(CFK), 163.5(DNR), Q164(M), 164.5(KPY), Q165(CHN), 165.5(FIPTV), P166(-ACFIKLNQRSTY), 166.5(FINTY), R168(K), R170(K), N172(-DSVW), 172.5(F), 173.5(DFHINQ), G174(ACDHQSTWY), 174.5(IK), Q175(ANRT), 175.5(I), T176(CG), 176.5(N), G177(-Q), D178(-IW), A179(K), 179.5(CY), D180(MNY), 180.5(M), 181.5(TY), 182.5(CLS), 183.5(M), 184.5(MY), P185(TW), 185.5(FQ), Q186(N), 186.5(CKY), P187(F), 187.5(QY), L188(MT), G189(IM), 189.5(MQ), Q190(MW), P192(CKM), 192.5(HM), 193.5(W), 194.5(M), S196(—), L198(VW), G199(D), 199.5(W), T200(N), M203(DEHW), T205(Y), G206(EKY), S207(D), G208(E), P210(CH), D213(HW), N214(F), N215(I), G217(DEFHITVY), A218(Q), G220(CMN), N223(DH), S225(CR), H229(ADV), T233(DHV), 237.5(HNQ), R238(H), 238.5(T), 1240(H), T242(HY), T246(I), T251(V), N254(C), L256(AGKMS), K258(Q), Q259(AGKPRS), 260.5(0), S261(ACGNT), 262.5(D), Q263(-DFGHLTWY), S264(-DEILQVWY), 265.5(ADENS), A266(S), 266.5(AE), S267(AENP), 267.5(E), N268(AEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(EINV), F273(HW), G280(A), F282(WY), D295(E), R307(-CDMY), P308(-N), L311(Y), K314(CFLQTVWY), I318(F), T324(L), 324.5(K), Q325(HKR), 325.5(R), D327(Q), T329(C), T330(K), T331(H), I332(CS), 332.5(K), A333(DHNY), L336(I), T339(C), V342(M), T344(FI), S346(F), Q349(AMS), F365(W), A367(P), L378(AV), T379(CG), 379.5(DE), L380(CEMNQTV), 380.5(E), N381(ACDGMQS), N382(CDEST), 382.5(D), 383.5(E), S384(G), 384.5(D), Q385(EKLTV), 385.5(K), A386(EHPTVW), 386.5(CDE), V387(CDETWY), 387.5(EKP), G388(M), R389(I), S390(ADGT), 395.5(W), Y397(AN), P399(Q), Q401(FIY), L403(C), R404(K), T405(SY), G406(DENQ), 406.5(Q), N408(DFGMQ), 408.5(N), F409(CH), T410(FM), S412(VW), 412.5(Q), Y413(M), T414(F), E416(G), 416.5(CE), D417(MN), 417.5(KM), V418(Q), P419(W), F420(LM), A425(FQ), H426(N), S427(AN), Q428(EHN), S429(GP), L430(MV), 431.5(Y), R432(-I), 433.5(F), L437(N), D439(KNSY), 444.5(CLM), L445(FMY), 445.5(ACDEGHILMNQSTV), S446(-EGHILMNTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-DEFGILMNPTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQSVWY), 448.5(ACDEFGHILMNPQY), N449(-DEHMPW), 449.5(AEHLNPS), T450(-CNP), 450.5(DEFGLT), P451(-I), 451.5(NP), S452(NP), 452.5(AL), G453(-AEFIKLNQST), 453.5(FINY), T454(DFHIN), 454.5(DFY), T455(-DEMNY), 455.5(HP), T456(DEFGHIN), 456.5(DEH), Q457(GP), 457.5(ADWY), S458(DEFW), 458.5(DEHILMNT), R459(-P), 459.5(DP), L460(HWY), 460.5(W), Q461(DFI), F462(Y), 462.5(N), S463(CGKNQV), 463.5(F), Q464(K), 464.5(SY), A465(G), 466.5(E), S468(CDEY), 469.5(E), R471(DEGQTV), D472(LMSTWY), Q473(F), S474(CI), R475(H), 475.5(SW), N476(ST), 476.5(L), W477(F), 477.5(L), 478.5(L), P479(GT), 479.5(D), G480(Y), P481(AV), 481.5(GHY), C482(GQWY), Y483(AT), 483.5(QV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(AGSY), 485.5(EIKM), Q486(ACDEFGILMPVWY), 486.5(FV), R487(-ACDEFGKLMNPQSTVWY), 487.5(FW), V488(AEFGHLMNQY), 488.5(ACDEFGILMPQSVWY), S489(-EFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVY), K490(-ACDFGPQWY), 490.5(CDFGHILMPQTVWY), T491(-GP), 491.5(AEFGHILMPQSTVWY), S492(-CGM), 492.5(AEGHILMQSTVY), A493(-HQ), 493.5(ADEHIKMNPQSTV), D494(FR), 494.5(ADFGHILMNPQSTVY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMNPQSTVWY), N496(-CDEFGILMPQTVW), 496.5(ADEFGHILMNPQSTVWY), N497(-CEFGHLMWY), 497.5(ADEFGILMNPQSTVY), S498(-CDEINPRTVW), 498.5(ADEFGHILMNPQSTVWY), E499(CIPS), 499.5(ACDEGILMNPQRSTVWY), Y500(-ACDEGHIKLNPQSTVW), 500.5(ADEFGHILMNPQSTVWY), S501(-CDFILPRVW), 501.5(ACDEFGHILMNPQRSTVWY), W502(-ACDEFGILMPQSVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGHLMNQSWY), 503.5(ADEFGILMNPQSTVW), G504(-CDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(FGILMPQ), 506.5(ACIPSTVW), K507(-EFNWY), Y508(IMV), H509(EFKNQR), 509.5(K), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(H), D514(ACFGILMV), S515(CINV), L516(ACS), V517(L), N518(V), P521(MS), M523(CV), 523.5(A), A524(G), 524.5(ACGHSTW), S525(CMNPT), 5255(ACDEFGHILMNPQSTVWY), H526(DEFGIKLMPRVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILPSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AEFMPQSVW), 528.5(DEPT), D529(ES), 529.5(ADEGLMNPQV), E530(DNPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGILMNPQSTVY), 531.5(ADEFGIKLMNPQRSTVY), K532(-CEP), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHIQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILVWY), 534.5(F), P535(-F), Q536(-ADFGHNPSTVWY), 536.5(ACILTV), S537(-DFGQY), 537.5(T), G538(—), V539(HL), L540(M), 541.5(D), 542.5(Q), 543.5(DIKPRT), K544(-F), 544.5(FKMRVWY), Q545(—), 545.5(CFIMNSTW), G546(CFHQ), 546.5(ACEGHMNQSW), S547(-DWY), 547.5(GHLNPSTWY), E548(AFGLP), 548.5(ACGHILNPT), K549(DQ), 549.5(ADLNQSTV), T550(-CY), 550.5(EKMPQRSTV), N551(-CPV), 551.5(ACEILMPQ), 552.5(V), D553(AGST), 1554(H), E555(GHNS), 556.5(Q), M558(IV), T560(V), D561(I), 565.5(Y), R566(DFHLQ), 567.5(E), P570(Q), 570.5(M), V571(ADEFHILMQSTY), 572.5(WY), T573(ACDGHIVWY), 573.5(T), E574(ACDFLMSW), Q575(CT), 575.5(WY), Y576(ACGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(FHLY), 578.5(ACDEFGHILNSTVY), V579(DFQWY), 579.5(ACDEFGILMNPQTVWY), S580(-DEFHILMNQVY), 580.5(ACDEFGKLMNPSTVY), T581(-GHPQW), 581.5(ACDEGILMNPQTVY), N582(-ACDEFGHIKLMPQRSTWY), 582.5(DFGHNPSTVW), L583(-DEGPWY), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMPQTVWY), R585(-ACDILMNPSTVWY), 585.5(DEFGHILMNPQSTVWY), G586(-HVY), 586.5(ADEFGHILMNPQSTVY), N587(-EFHILVY), 587.5(ADEFGHILMNPQSTVWY), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEFGHLMNPSY), Q589(-DEFGINPRSTVY), 589.5(ACDEFGHIKLMNPSTVWY), A590(DEFHKLMNQTWY), 590.5(ACDEFGIKLMPQSTVWY), A591(-FPWY), 591.5(ACEFGHILMNPQSTVY), T592(-CEGHLMP), 592.5(ACDEFGILMNPQSTVWY), A593(-FPY), 593.5(ACDEFGHILMNPQSTVWY), D594(LT), 594.5(ACDEFGHILMNPQSTVWY), V595(DEFHNPQWY), 595.5(ACDEFGHILMNQSTVW), N596(EFIPQW), 596.5(DEY), T597(P), 597.5(F), Q598(EGN), G599(W), 600.5(F), L601(W), P602(CQ), M604(C), 606.5(P), D608(W), D610(Y), 610.5(F), 613.5(Y), Q614(T), I617(K), 617.5(1), K620(CMN), H627(M), H629(WY), P630(G), L633(M), M634(C), F637(I), K640(VW), 640.5(N), Q645(M), I646(M), L647(I), N650(C), V653(C), N656(Q), 657.5(C), 658.5(ACHN), 659.5(CDI), T660(HM), 660.5(E), F661(AEILQST), 661.5(CGKPRY), 662.5(CKMQW), 663.5(CGPW), A664(KW), 664.5(CMPR), K665(H), 665.5(HINRTWY), F666(C), 666.5(1), A667(CM), 667.5(QY), T671(R), S674(G), S679(FGHIKRY), V680(C), E681(D), Q687(I), K688(P), E697(D), 697.5(G), 1698(D), 699.5(E), 702.5(Q), N703(G), 703.5(AEHLTY), 704.5(AILNS), 705.5(QY), K706(DEST), S707(CEHILMNY), 707.5(MY), V708(ACGHN), 708.5(1), N709(P), 709.5(GPQ), V710(AMNPQ), 710.5(D), D711(GN), T713(C), T716(CKRW), N717(R), V719(KY), S721(CHKLMNR), E722(Q), P723(CDEN), R724(FSY), P725(I), I726(L) 1.5(MV), A2(CS), 2.5(GIMP), A3(CDEFI), 3.5(ILTVY), D4(CS), 0.7-fold 4.5(AFHNVY), G5(-CN), 5.5(CFKM), 6.5(N), WIO(ACEGIKPQRSTY), 10.5(HI), L11(ACV), 11.5(K), E12(FHIW), D13(CEGIKVWY), T14(CDFHIPQR), L15(HTW), E17(CFINQRTVY), G18(ADS), I19(AEGHNQST), R20(FILQV), Q21(GHKTV), 21.5(FWY), W22(FIV), 22.5(FILMWY), W23(CDEHKMNQ), 23.5(ACDEFGHLNQSTWY), 24.5(CDEHILNY), L25(AEFGHNQRS), 25.5(CEIKLNQRSTVWY), K26(DHNY), 26.5(HNQ), P27(T), 27.5(G), 28.5(AEFGILNPQSTVWY), P29(DEFGLQRSTW), P30(ADFHQRSY), 30.5(ADEFGIKLNPQRSTY), P31(DHK), 31.5(AFILPV), P32(AEGHILNQRSTY), 32.5(ADEKLNPRSTW), K33(-DGNPQRSY), 33.5(CEFKNPQTVWY), P34(-GLQRTVY), 34.5(CEFGHKQRVY), A35(EGHKNQVY), 35.5(CGHT), E36(FHK), 36.5(FINW), R37(-DN), 37.5(MPQ), H38(CENW), 38.5(CDEGIKNP), K39(AFLVY), 39.5(CDGIY), D40(-ACNY), 40.5(CDGY), D41(EHQRS), 41.5(DFNRTVW), S42(-DKLQRTVW), 42.5(CDGT), R43(ASTV), 43.5(DPRV), 45.5(M), V46(I), L47(F), P48(Y), G49(CFLMQY), Y50(HW), 50.5(N), K51(ACFLMNVY), Y52(D), 53.5(R), P55(Y), 55.5(EKY), 56.5(K), G58(CES), 58.5(F), L59(ACFGKQRW), 59.5(H), D60(CHNTY), K61(-CILQS), 61.5(GK), G62(Q), E67(DLQ), A68(LT), L73(EHKV), E74(QTW), K77(D), 77.5(K), A78(FY), 78.5(ADFHIQR), Y79(-C), 79.5(CK), 80.5(F), R81(CDQ), 8E5(FHW), Q82(EHMN), 82.5(DIK), L83(CGN), 83.5(FGHIKLQWY), 84.5(ENQ), S85(-E), 85.5(GHKS), G86(-ACDEVW), 86.5(FQSVW), D87(ACHK), 87.5(ADEFGQ), N88(A), P89(CDH), 89.5(GL), 90.5(EMY), L91(Q), K92(G), 92.5(RY), Y93(GHNW), 93.5(Y), 94.5(AY), H95(AEGRY), A96(QVW), D97(-G), A98(CDW), 98.5(CE), E99(CG), 99.5(N), F100(K), Q101(DVY), 101.5(E), E102(FQ), R103(IN), 103.5(SV), L104(NT), 104.5(ADEGKNV), K105(FMQW), 105.5(CQRWY), E106(CFIK), 106.5(EFHNRY), D107(-CFKVY), 107.5(NP), T108(-CI), 108.5(IQY), S109(CILN), 109.5(AHY), F110(CEHNY), G111(CE), 11L5(AW), G112(P), 112.5(CQ), N113(CDHIKLQRVW), 113.5(FHN), L114(FN), G115(-DEFMPV), 115.5(ADEHLNVW), R116(EGMP), 116.5(ADEGHIKLPQTWY), A117(CEHKQRWY), 117.5(DEFKLPQW), V118(-DKPRW), 118.5(ACEGMP), F119(ACHQS), 119.5(AHIKLNQRSTVY), Q120(-FKW), 120.5(GKLPR), A121(EFKNQ), 121.5(FGHQ), K122(-DFGNRW), 122.5(DFHIKPQSW), K123(CDFHT), 123.5(AFKNPQT), R124(-CHIP), 124.5(CEFHKPRS), V125(-CDEHPQSW), 125.5(ACEFGQRSW), L126(-KQR), 126.5(CER), E127(-P), 127.5(AGILNSTY), P128(CDFILQRSTV), 128.5(AGHKLRSTW), L129(DKR), 129.5(CFQ), G130(EFQ), 130.5(CEHKLSTY), L131(DI), 131.5(ADEFHLP), V132(CR), 132.5(HIKLNVWY), E133(GLS), 133.5(PW), 134.5(HW), P135(H), 135.5(LW), V136(-HY), 136.5(AEHMNQ), K137(-CDPTY), 137.5(CDGHIKLNPQRSVWY), T138(AEGPQ), 138.5(CDEGKNQSW), A139(-CDEFKQTW), 139.5(CDGHRY), P140(DEGHTW), 140.5(CDEKNQ), G141(DEFHIKLMNQRTWY), 141.5(DEHKQRTVY), K142(-CDEGNPY), 142.5(CDEFGIKNPQSWY), K143(EGRVY), 143.5(AEFKLPQSTY), R144(HNPSTVW), 144.5(ACEFHILNPQRSTVWY), P145(-ADEFGILNSVWY), 145.5(DEGKMPQTV), V146(DEKQRTW), 146.5(CEFKLNPRY), E147(FGHKLNQVY), 147.5(EFHK), H148(CGW), 148.5(HKMNR), S149(-ACEHIMNPQR), 149.5(CHIKMNRTW), P150(DFHMR), 150.5(DEMPRW), V151(-CDGY), 151.5(DY), E152(CFP), 152.5(DGHKNY), D154(S), 155.5(D), S156(AT), S157(EFKRTV), 157.5(DNQTW), G158(D), 158.5(F), T159(EFGHPQ), 159.5(Y), G160(Y), 160.5(DNWY), K161(GHNTVY), 161.5(FGILQRSV), A162(IQ), 162.5(EPRT), G163(CFKN), 163.5(DEKNRSTWY), Q164(CMR), 164.5(DEFGIKNPRY), Q165(CHILNVW), 165.5(FIPTV), P166(-ACFHIKLNQRSTY), 166.5(FGHINPSTY), A167(QSV), R168(K), R170(K), N172(-ACDFHLQSVW), 172.5(F), 173.5(ADFHINQWY), G174(ACDEFHQSTWY), 174.5(IKNS), Q175(-ADNRST), 175.5(AFGIKNY), T176(ACGY), 176.5(ENP), G177(-Q), 177.5(EGI), D178(-EIWY), 178.5(INPS), A179(-IKW), 179.5(CDHNPQRSTVWY), D180(-ACFIMNY), 180.5(CFHIMQ), S181(FKL), 181.5(CGHNTVY), 182.5(CHLPS), P183(CS), 183.5(EGKMNY), D184(FGHINPQR), 184.5(MPRY), P185(DEFILNTVWY), 185.5(CFIPQTVW), Q186(FNPTY), 186.5(CDEKSY), P187(FGIL), 187.5(CGKPQWY), L188(EFIMSTW), 188.5(DN), G189(CIMN), 189.5(MQW), Q190(MW), 190.5(CFGH), P191(N), 191.5(CIQW), P192(CKMWY), 192.5(EHMY), A193(DNY), 193.5(DHMRVW), A194(CFGIQV), 194.5(CFMP), P195(DKQRSTY), 195.5(DHMQST), S196(-FK), L198(DVWY), G199(D), 199.5(FHW), T200(AFINQR), N201(CH), T202(KPSV), M203(ACDEFGHIPRTVWY), T205(FKRVY), G206(ACDEHKRY), S207(ADRY), G208(EFHKLQWY), A209(DEMY), P210(CHR), D213(ACFHIKLNTW), N214(ACEFLVY), N215(CHILY), E216(AH), G217(ACDEFHILMNSTVY), A218(Q), G220(ACMNT), N223(ACDHIY), S225(CQR), H229(ADSTV), T233(DHIQVY), M235(L), D237(C), 237.5(EHKNQ), R238(HS), 238.5(TY), 1240(H), T242(CHRY), T246(I), P250(S), T251(SV), N254(CQS), L256(ACGKMQRSV), K258(Q), Q259(AGHKPRS), 260.5(MQ), S261(ACGNT), 262.5(D), Q263(-DEFGHLTWY), S264(-DEILQVWY), 265.5(ADEMNQS), A266(S), 266.5(AE), S267(AENP), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(EFINV), F273(HWY), S276(K), G280(A), F282(LWY), F284(CY), F287(Y), H288(N), S292(C), D295(AE), R307(-CDKMSY), 307.5(FK), P308(-NV), K309(AQ), R310(GILV), L311(CFY), N312(HMT), K314(CFGLQSTVWY), L315(V), 1318(F), T324(LM), 324.5(KR), Q325(HKRV), 325.5(FHIKR), N326(DMRWY), D327(QT), G328(AN), T329(CIY), T330(KQ), T331(FHY), I332(ACS), 332.5(K), A333(DFHNTY), N334(S), L336(I), T339(C), V342(ACIM), T344(DFILN), S346(CFHLMPQW), Y348(FRS), Q349(AGMS), V353(CI), S356(A), H358(ACGN), Q359(M), F365(W), A367(CP), V369(PT), M371(CIKQS), G376(CS), L378(ACV), T379(ACGSV), 379.5(DE), L380(CEMNQTVY), 380.5(E), N381(ACDGMQS), N382(CDEST), 382.5(D), 383.5(E), S384(AG), 384.5(D), Q385(EKLMSTV), 385.5(DEK), A386(-EHLNPRTVW), 386.5(CDEY), V387(CDEQTWY), 387.5(EGKP), G388(EIM), R389(IL), S390(ADGNT), C394(V), 394.5(N), 395.5(W), Y397(AGN), P399(EIMQR), Q401(CDFIKLNRVWY), L403(C), R404(K), 404.5(N), T405(SY), G406(DENQ), 406.5(Q), 407.5(H), N408(ADFGHLMQTY), 408.5(N), F409(CH), T410(CFHLMY), F411(CTW), S412(IKVWY), 412.5(Q), Y413(FM), T414(FK), 414.5(D), 415.5(APQ), E416(DG), 416.5(CEMR), D417(AIKMNTV), 417.5(DKLMRS), V418(GQ), P419(EINTVW), F420(LM), S422(A), S423(LT), Y424(FH), A425(-CFQV), H426(N), S427(AN), Q428(EHN), S429(CGHPY), L430(MV), D431(M), 431.5(Y), R432(-I), L433(V), 433.5(F), L437(NS), I438(M), D439(KNPSY), 441.5(M), 444.5(CLM), L445(FMY), 445.5(ACDEGHILMNQSTVY), S446(-DEGHILMNQTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-ADEFGILMNPQTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQSVWY), 448.5(ACDEFGHILMNPQSY), N449(-CDEHILMPVWY), 449.5(AEGHLMNPST), T450(-CFGNPY), 450.5(ADEFGHLTV), P451(-I), 451.5(NP), S452(-DEFGNPQTY), 452.5(ALV), G453(-AEFIKLMNPQST), 453.5(FILNY), T454(-DEFGHILMNP), 454.5(DEFPY), T455(-ADEILMNPQVY), 455.5(HNP), T456(DEFGHIKMNS), 456.5(DEGHIL), Q457(EGHILMPS), 457.5(ADEGHNTWY), S458(DEFHMNW), 458.5(CDEGHILMNT), R459(-EGINPY), 459.5(DEP), L460(AHIMTWY), 460.5(W), Q461(DFHIKV), F462(Y), 462.5(N), S463(ACDGKMNQRV), 463.5(F), Q464(IKLMV), 464.5(SY), A465(G), G466(A), 466.5(E), A467(EM), S468(CDELPQVY), D469(V), 469.5(E), R471(DEGQSTV), D472(GHLMSTVWY), Q473(FHY), S474(CIY), R475(H), 475.5(SW), N476(ST), 476.5(L), W477(F), 477.5(L), L478(FI), 478.5(L), P479(GIT), 479.5(D), G480(Y), P481(ACV), 48E5(GHY), C482(AFGQSWY), Y483(AITW), 483.5(FHQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACGNSTY), 485.5(EIKMY), Q486(ACDEFGILMPSVWY), 486.5(FV), R487(-ACDEFGIKLMNPQSTVWY), 487.5(FW), V488(AEFGHILMNQTWY), 488.5(ACDEFGILMPQSVWY), S489(-EFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGMNPQRSTVWY), 490.5(CDEFGHILMPQRSTVWY), T491(-AGPY), 491.5(ADEFGHILMNPQSTVWY), S492(-CFGLMPQT), 492.5(AEGHILMPQSTVY), A493(-HQ), 493.5(ADEGHIKLMNPQSTVY), D494(EFR), 494.5(ADFGHILMNPQSTVWY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMNPQSTVWY), N496(-ACDEFGILMPQTVWY), 496.5(ADEFGHILMNPQSTVWY), N497(-CEFGHLMVWY), 497.5(ADEFGILMNPQSTVY), S498(-CDEINPRTVW), 498.5(ADEFGHILMNPQSTVWY), E499(CIPS), 499.5(ACDEGILMNPQRSTVWY), Y500(-ACDEGHIKLNPQSTVW), 500.5(ADEFGHILMNPQSTVWY), S501(-CDEFILMPRTVW), 501.5(ACDEFGHILMNPQRSTVWY), W502(-ACDEFGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGHLMNQSWY), 503.5(ADEFGILMNPQSTVW), G504(-ACDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-FGILMPQS), 506.5(ACIPSTVW), K507(-ACEFHLNVWY), Y508(-DIMSTV), H509(EFKNQR), 509.5(K), L510(I), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(HI), D514(ACFGILMSV), S515(ACINTV), L516(ACMRS), V517(L), N518(TVY), P521(FMS), 521.5(M), A522(CV), M523(CLV), 523.5(A), A524(GP), 524.5(ACGHSTW), S525(CMNPT), 525 5(ACDEFGHILMNPQSTVWY), H526(DEFGIKLMPRVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILPSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AEFMPQSVW), 528.5(DEPT), D529(ES), 529.5(ADEGLMNPQTV), E530(DNPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGHILMNPQSTVY), 531.5(ADEFGIKLMNPQRSTVY), K532(-CDEP), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHILNQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILVWY), 534.5(F), P535(-AF), Q536(-ACDEFGHMNPSTVWY), 536.5(ACILTV), S537(-DFGNQTY), 537.5(T), G538(—), V539(HLMQST), L540(M), 540.5(N), I541(DERT), 541.5(ACDQV), 542.5(Q), G543(APS), 543.5(DHIKLMPRTV), K544(-FS), 544.5(AFGHIKLMNRVWY), Q545(-AKLRW), 545.5(ACFHILMNPQSTVWY), G546(ACEFHLPQSVWY), 546.5(ACDEGHILMNQSTVWY), S547(-DGLNQWY), 547.5(ADGHILMNPSTVWY), E548(AFGLMNPS), 548.5(ACDGHILNPSTWY), K549(-DEFGHLPQSY), 549.5(ADFGLMNQSTVY), T550(-CQY), 550.5(AEGIKMPQRSTV), N551(-CEGIPQRSV), 551.5(ACDEFILMPQST), V552(CQS), 552.5(VW), D553(AGST), I554(AHTW), E555(AGHNSTVY), K556(CDM), 556.5(Q), V557(IL), M558(IV), I559(FQV), T560(V), D561(INRVY), I565(T), 565.5(Y), R566(DFHLQY), 567.5(E), T568(P), 569.5(M), P570(QS), 570.5(M), V571(ADEFHILMQSTY), A572(CY), 572.5(WY), T573(ACDGHINSVWY), 573.5(T), E574(ACDFLMSW), 574.5(V), Q575(-CFHILTV), 575.5(AWY), Y576(ACFGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(ACFHLY), 578.5(ACDEFGHILNSTVY), V579(DFGLMNQSWY), 579.5(ACDEFGILMNPQSTVWY), S580(-DEFHILMNPQTVY), 580.5(ACDEFGKLMNPQSTVWY), T581(-FGHLNPQWY), 581.5(ACDEFGILMNPQTVY), N582(-ACDEFGHIKLMPQRSTVWY), 582.5(DFGHNPSTVW), L583(-ADEFGMPSWY), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMPQSTVWY), R585(-ACDFGILMNPSTVWY), 585.5(ADEFGHILMNPQSTVWY), G586(-DEHMVY), 586.5(ADEFGHILMNPQSTVY), N587(-EFHILMPTVY), 587.5(ADEFGHILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(ADEFGHLMNPSTVY), Q589(-ADEFGINPRSTVWY), 589.5(ACDEFGHIKLMNPQSTVWY), A590(DEFHKLMNQTWY), 590.5(ACDEFGIKLMPQSTVWY), A591(-FILNPVWY), 5 91.5(ACDEFGHILMNPQSTVY), T592(-CDEGHLMNPQVY), 592.5(ACDEFGILMNPQSTVWY), A593(-FLMNPWY), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(ACDEFGHMNPQSTWY), 595.5(ACDEFGHILMNQSTVW), N596(ADEFGHIMPQTWY), 596.5(DENY), T597(FKP), 597.5(F), Q598(AEGHN), G599(SW), V600(M), 600.5(F), L601(VW), P602(CMQ), M604(CS), V605(AFN), 605.5(N), 606.5(P), D608(W), R609(D), D610(Y), 610.5(F), 613.5(Y), Q614(T), G615(S), P616(AC), I617(KM), 617.5(I), K620(ACGHMN), I621(K), H623(CDFLW), H627(MNQ), F628(L), H629(WY), P630(AGQ), 630.5(C), L633(HM), M634(C), F637(I), L639(FV), K640(ANTVW), 640.5(N), H641(CIK), Q645(LMT), I646(MT), L647(IY), N650(CILV), V653(CT), 655.5(M), N656(QW), 657.5(CHSVW), 658.5(ACEHIKNQR), T659(-CQ), 659.5(CDFGIMY), T660(-DHKMQ), 660.5(EFHIKPQWY), F661(AEILMQSTV), 661.5(ACEFGIKLMPRSVY), S662(CPW), 662.5(ACDFKMPQRVW), A663(KLNY), 663.5(CGIMNPRSVWY), A664(KWY), 664.5(ACMPRTWY), K665(CFGHM), 665.5(HILNPQRSTWY), F666(CGWY), 666.5(IV), A667(CIMY), 667.5(QY), I670(V), T671(NRS), Q672(CEGLS), S674(G), V678(C), S679(CFGHIKLRY), V680(CL), E681(DLN), E683(H), E685(L), Q687(IV), K688(HP), N690(R), E697(DI), 697.5(GHNPW), I698(DL), 699.5(E), T701(S), 701.5(A), S702(QV), 702.5(Q), N703(DG), 703.5(AEHLTY), Y704(H), 704.5(ADEILNST), N705(CDPV), 705.5(ENQY), K706(DEGRST), S707(CDEGHILMNQTVY), 707.5(MY), V708(ACEGHNRS), 708.5(I), N709(KP), 709.5(GPQ), V710(AELMNPQ), 710.5(D), D711(GN), 711.5(Q), T713(CS), T716(CIKNQRWY), N717(ERS), V719(KNQY), 720.5(N), S721(CFHIKLMNRVW), E722(DQ), P723(ACDENQSV), R724(FHKSWY), P725(ACIQ), I726(LV) 1.5(MV), A2(CS), 2.5(AGIMP), A3(CDEFILQ), 3.5(ADFILTVY), 0.8-fold D4(ACSTV), 4.5(AFGHNVY), G5(-CN), 5.5(CFKM), 6.5(N), D9(A), W10(ACEGIKLPQRSTY), 10.5(HI), L11(ACV), 11.5(K), E12(DFGHILSW), D13(CEGIKLRVWY), T14(CDFGHINPQR), L15(AHTW), E17(CFINQRSTVY), G18(ADS), I19(AEGHNQSTV), R20(FILQSV), Q21(FGHIKNRTVY), 21.5(FWY), W22(FIVY), 22.5(FILMWY), W23(CDEHKLMNQRSTV), 23.5(ACDEFGHLNQSTWY), K24(ALNPQ), 24.5(CDEHILNY), L25(AEFGHNQRST), 25.5(CEGHIKLNQRSTVWY), K26(DHNY), 26.5(HNQ), P27(T), 27.5(G), 28.5(AEFGILNPQSTVWY), P29(DEFGHILQRSTW), P30(ADFHQRSY), 30.5(ADEFGIKLNPQRSTY), P31(DHKLNY), 31.5(AFILPV), P32(AEGHIKLNQRSTY), 32.5(ADEIKLNPRSTWY), K33(-DFGNPQRSTWY), 33.5(CDEFHIKNPQTVWY), P34(-AGKLNQRTVY), 34.5(CEFGHKQRSVY), A35(EGHKLNQVY), 35.5(CEFGHINPTV), E36(CFGHKW), 36.5(DFHIKNW), R37(-DNSW), 37.5(DFMPQW), H38(-CDENVWY), 38.5(ACDEGIKNPR), K39(AFILRVWY), 39.5(CDGIY), D40(-ACGNY), 40.5(CDGSTY), D41(ACEHQRS), 41.5(CDFNRTVW), S42(-DKLQRTVWY), 42.5(ACDGKTY), R43(ASTV), 43.5(DPRV), 45.5(M), V46(I), L47(F), P48(LY), G49(CFLMQY), Y50(HW), 50.5(N), K51(ACFLMNVY), Y52(D), 53.5(R), P55(Y), 55.5(EKY), 56.5(K), G58(CES), 58.5(F), L59(ACFGKQRW), 59.5(H), D60(ACEHNQTY), K61(-ACILQRS), 61.5(GK), G62(Q), E63(HMN), V65(H), E67(CDLQW), A68(LT), A70(Y), A71(GKWY), L73(CEHKVY), E74(LQTVW), K77(CDTY), 77.5(K), A78(FNRY), 78.5(ADFHIQR), Y79(-C), 79.5(CK), D80(AMY), 80.5(F), R81(CDGQ), 81.5(FHW), Q82(-CEGHIMN), 82.5(DIK), L83(ACFGKN), 83.5(EFGHIKLQTVWY), D84(LQS), 84.5(ENQ), S85(-EPW), 85.5(AGHKSY), G86(-ACDESVW), 86.5(FGQSVW), D87(ACFGHKRY), 87.5(ADEFGQY), N88(AHLR), P89(CDH), 89.5(GL), 90.5(EMY), L91(QR), K92(GIRSV), 92.5(RY), Y93(FGHNW), 93.5(Y), N94(W), 94.5(AY), H95(ACDEGLRSY), A96(QVW), D97(-G), A98(CDWY), 98.5(CE), E99(CG), 99.5(KN), F100(KY), Q101(ADPVY), 101.5(E), E102(FLQY), R103(DINY), 103.5(SV), L104(INT), 104.5(ADEGKNV), K105(FMQRW), 105.5(CGLQRVWY), E106(CFIKY), 106.5(EFHNRVY), D107(-CFKNQTVWY), 107.5(NP), T108(-CINPY), 108.5(IQTY), S109(ACFIKLN), 109.5(AHY), F110(ACEHINY), G111(CE), 111.5(AW), G112(AP), 112.5(CQ), N113(CDGHIKLQRTVW), 113.5(FHN), L114(CFGN), G115(-DEFMPSV), 115.5(ADEHLNVW), R116(EGMP), 116.5(ADEGHIKLPQRSTVWY), A117(CEHKQRWY), 117.5(DEFKLPQW), V118(-DKLPRWY), 118.5(ACEGMP), F119(ACHLQSVWY), 119.5(AHIKLNQRSTVY), Q120(-CFIKRW), 120.5(GKLPR), A121(-EFGHKNQW), 121.5(FGHQR), K122(-DFGNRW), 122.5(ADFHIKPQSW), K123(CDEFHQSTY), 123.5(AFKNPQT), R124(-CGHIPY), 124.5(CEFHKPRS), V125(-ACDEHNPQSW), 125.5(ACEFGQRSW), L126(-CEKQRWY), 126.5(CERS), E127(-HIMPWY), 127.5(ADGILNSTY), P128(ACDFILQRSTV), 128.5(AGHKLRSTW), L129(CDGKRWY), 129.5(CDFGQRY), G130(CEFQ), 130.5(CDEHKLQRSTY), L131(DIQVY), 131.5(ACDEFHLPW), V132(-CDRS), 132.5(EHIKLNPRVWY), E133(-GHLS), 133.5(GIPW), E134(-W), 134.5(EHIQW), P135(CDHY), 135.5(DELNPRWY), V136(-DHQY), 136.5(ACEHILMNPQRTVY), K137(-CDHLPRSTWY), 137.5(ACDEGHIKLNPQRSTVWY), T138(AEGHKNPQR), 138.5(CDEFGKNQSTW), A139(-CDEFIKLPQSTWY), 139.5(ACDGHNRVY), P140(ADEFGHKTW), 140.5(CDEKNQRV), G141(-CDEFHIKLMNQRSTVWY), 141.5(DEFHIKLQRTVY), K142(-ACDEFGINPSY), 142.5(ACDEFGHIKNPQSTVWY), K143(EGIRSVY), 143.5(AEFKLPQRSTWY), R144(AHNPSTVW), 144.5(ACEFGHIKLNPQRSTVWY), P145(-ADEFGIKLNRSTVWY), 145.5(DEGKMNPQRSTVW), V146(-ACDEKLNQRTWY), 146.5(ACEFHIKLNPQRVY), E147(AFGHKLNQTVY), 147.5(EFHIKRS), H148(CGRTW), 148.5(AHKMNPQR), S149(-ACEGHIMNPQRW), 149.5(ACGHIKLMNRSTW), P150(DFHMQR), 150.5(CDEKLMNPQRTVWY), V151(-CDGKWY), 151.5(DWY), E152(CFNPV), 152.5(DGHKNRY), D154(S), 155.5(D), S156(AT), S157(EFKRTV), 157.5(DNQTW), G158(D), 158.5(F), T159(DEFGHPQR), 159.5(Y), G160(CY), 160.5(DFHNWY), K161(-GHLNQSTVY), 161.5(ADFGILQRSTVW), A162(DIKQ), 162.5(ACENPQRST), G163(-CFHKNRV), 163.5(DEKLNPRSTVWY), Q164(CHIMRVW), 164.5(DEFGIKNPQRSY), Q165(CHILNPVW), 165.5(FIPQTV), P166(-ACFGHIKLNQRSTY), 166.5(FGHINPSTY), A167(QSV), R168(K), R170(K), L171(I), N172(-ACDFHLQSVW), 172.5(F), 173.5(ADEFGHINQWY), G174(ACDEFHQSTWY), 174.5(AEFHIKNRSY), Q175(-ACDGNRST), 175.5(AFGIKLNQY), T176(ACDFGY), 176.5(ADENP), G177(-EQ), 177.5(EGIT), D178(-EISWY), 178.5(FINPRS), A179(-IKPSW), 179.5(CDHKNPQRSTVWY), D180(-ACFIMNQVY), 180.5(ACFHILMQR), S181(FKLTY), 181.5(ACGHNSTVY), V182(ACIS), 182.5(CHLPRS), P183(CHRS), 183.5(EGKMNQY), D184(CFGHINPQRTY), 184.5(CLMPRY), P185(CDEFGILNTVWY), 185.5(CFILPQTVW), Q186(FIKNPTY), 186.5(CDEIKNSY), P187(-FGILNQS), 187.5(CEGKNPQWY), L188(CEFIMQSTW), 188.5(ADNQ), G189(-CIMN), 189.5(FLMQW), Q190(CMNRW), 190.5(ACFGHQR), P191(KN), 191.5(CIQVW), P192(-CDFKMSWY), 192.5(EGHLMVY), A193(CDGHILNY), 193.5(DHIMRTVW), A194(CFGIQVW), 194.5(CFKMPT), P195(DEGHKQRSTVY), 195.5(CDHMQST), S196(-FKNPRT), G197(A), L198(ACDFVWY), G199(D), 199.5(FHRW), T200(ACFGINQR), N201(CDH), T202(KPQRSV), M203(ACDEFGHILPQRTVWY), A204(DV), T205(FKMRVWY), G206(ACDEFHKQRY), S207(ACDRY), G208(AEFHKLNQRWY), A209(DEKMRSY), P210(CGHR), D213(ACFGHIKLNTW), N214(ACDEFKLPSVY), N215(CHILY), E216(ADH), G217(ACDEFHILMNSTVY), A218(QST), G220(ACMNT), V221(I), N223(ACDHIMQSTY), S225(CQR), H229(ADSTV), D231(C), T233(DHIMQSVY), M235(LV), 236.5(D), D237(C), 237.5(DEHKNQ), R238(CHS), 238.5(CFTY), 1240(H), T242(CFHRY), S243(A), T246(I), L249(C), P250(AQS), T251(CLSV), N253(D), N254(CQS), H255(A), L256(ACGKMQRSV), K258(LQ), Q259(AGHKPRS), 260.5(MQ), S261(ACGNT), 262.5(D), Q263(-DEFGHLTWY), S264(-ADEILQTVWY), 264.5(D), 265.5(ADEMNQST), A266(S), 266.5(AE), S267(AENP), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(EFINV), F273(HWY), S276(K), G280(A), Y281(H), F282(LWY), F284(CY), F287(Y), H288(N), S292(C), D295(AE), N303(TV), F306(W), R307(-CDKMSTY), 307.5(FKN), P308(-NV), K309(AQS), R310(AGILVW), L311(CFY), N312(DHLMQTWY), K314(CFGHLMQSTVWY), L315(V), I318(F), T324(LMN), 324.5(CKR), Q325(CHKRV), 325.5(FGHIKRST), N326(CDMRWY), 326.5(N), D327(QT), 327.5(GHT), G328(AN), 328.5(G), T329(ACIY), T330(KQS), T331(FHY), I332(ACS), 332.5(K), A333(DFHNTY), N334(S), L336(I), T339(C), V342(ACIM), T344(DFIKLMNV), S346(CFHLMPQW), Y348(FNRS), Q349(AGMS), V353(CI), L354(QT), G355(S), S356(A), H358(ACGNST), Q359(DEHM), C361(A), F365(W), A367(CP), V369(PT), M371(CIKLNQS), V372(CI), Y375(W), G376(CS), Y377(F), L378(ACV), T379(ACGSV), 379.5(DE), L380(CEMNQTVY), 380.5(E), N381(ACDGMQS), N382(CDEST), 382.5(D), 383.5(E), S384(AG), 384.5(D), Q385(ACEIKLMRSTVY), 385.5(DEKLPT), A386(-EHLNPRSTVW), 386.5(CDEY), V387(CDEQTWY), 387.5(EGKP), G388(EIM), R389(GIL), S390(ADGNT), 390.5(C), S391(A), C394(V), 394.5(N), 395.5(W), Y397(AGN), P399(EIMQRS), Q401(ACDEFIKLNRVWY), L403(C), R404(K), 404.5(N), T405(SY), G406(DENQ), 406.5(Q), 407.5(H), N408(ADFGHLMQSTWY), 408.5(CN), F409(CHW), T410(CEFHLMQVY), F411(CLTVW), S412(FGIKMVWY), 412.5(Q), Y413(FM), T414(EFIKLNQ), 414.5(D), 415.5(AGNPQS), E416(ADG), 416.5(CEKMNR), D417(AIKMNTV), 417.5(DEKLMQRS), V418(GMQS), P419(EIKNSTVW), F420(LM), S422(A), S423(CLT), Y424(FHT), A425(-CFQV), H426(N), S427(AN), Q428(EHN), S429(CGHPY), L430(MV), D431(M), 431.5(Y), R432(-I), L433(IV), 433.5(F), M434(A), L437(ANSV), I438(GHM), D439(KNPSTY), 441.5(M), L442(ITV), 444.5(CLM), L445(CFIMY), 445.5(ACDEGHILMNQSTVY), S446(-DEGHILMNQTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-ACDEFGHILMNPQSTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQSVWY), 448.5(ACDEFGHILMNPQSY), N449(-CDEFHILMPTVWY), 449.5(AEGHLMNPST), T450(-CFGNPSY), 450.5(ADEFGHLQTV), P451(-IL), 451.5(NP), S452(-DEFGLNPQTY), 452.5(ALV), G453(-AEFHIKLMNPQST), 453.5(FILNY), T454(-DEFGHILMNPSV), 454.5(ADEFGMPY), T455(-ADEILMNPQSVY), 455.5(FHNPQ), T456(DEFGHIKLMNQSY), 456.5(DEGHILPS), Q457(EGHILMPS), 457.5(ADEFGHLMNPTWY), S458(-DEFHMNVW), 458.5(CDEGHILMNTVW), R459(-DEGINPY), 459.5(DEP), L460(AHIMPTWY), 460.5(W), Q461(DFHIKMV), F462(Y), 462.5(N), S463(ACDGKMNQRTV), 463.5(F), Q464(IKLMRTV), 464.5(SY), A465(G), G466(AD), 466.5(E), A467(EMS), S468(ACDEHLNPQVWY), D469(V), 469.5(E), R471(DEGQSTV), D472(GHLMQSTVWY), Q473(FHSY), S474(ACIY), R475(HK), 475.5(SW), N476(CSTW), 476.5(L), W477(F), 477.5(L), L478(FI), 478.5(L), P479(GIT), 479.5(D), G480(Y), P481(ACV), 481.5(GHY), C482(AFGQSWY), 482.5(F), Y483(AHITVW), 483.5(FHQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACFGILNSTY), 485.5(EIKMY), Q486(ACDEFGILMPSVWY), 486.5(FV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(ACEFGHILMNQTWY), 488.5(ACDEFGILMPQSVWY), S489(-ACEFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACDEFGHILMPQRSTVWY), T491(-AGPSY), 491.5(ADEFGHILMNPQSTVWY), S492(-CFGHLMPQTV), 492.5(ADEGHILMPQSTVY), A493(-HQTV), 493.5(ADEFGHIKLMNPQSTVY), D494(EFR), 494.5(ADEFGHILMNPQSTVWY), N495(-ADEFGHILMPQSTVWY), 495 5(ACDEFGILMNPQSTVWY), N496(-ACDEFGILMPQTVWY), 496.5(ADEFGHILMNPQSTVWY), N497(-CEFGHLMVWY), 497.5(ADEFGILMNPQSTVY), S498(-CDEINPQRTVW), 498.5(ADEFGHILMNPQSTVWY), E499(CIPS), 499.5(ACDEGILMNPQRSTVWY), Y500(-ACDEGHIKLNPQSTVW), 500.5(ADEFGHILMNPQSTVWY), S501(-CDEFGILMNPRTVWY), 501 5(ACDEFGHILMNPQRSTVWY), W502(-ACDEFGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGHLMNPQSWY), 503.5(ADEFGILMNPQSTVW), G504(-ACDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-FGILMNPQS), 506.5(ACIPSTVW), K507(-ACEFHLMNVWY), Y508(-DIMSTV), H509(EFKNQR), 509.5(K), L510(I), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(HI), D514(ACFGILMQSTV), S515(ACINTV), L516(ACIMRS), V517(CL), 517.5(C), N518(AILQTVY), P521(CFMS), 521.5(M), A522(CSV), M523(CLV), 523.5(A), A524(GP), 524.5(ACGHSTW), S525(CMNPT), 525.5(ACDEFGHILMNPQSTVWY), H526(-DEFGIKLMPRVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILPSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AEFMPQSVW), 528.5(DEPT), D529(ES), 529.5(ADEGLMNPQTV), E530(DNPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGHILMNPQSTVY), 531.5(ADEFGIKLMNPQRSTVY), K532(-ACDEPR), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHILNQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-ACDEFGHIMNPSTVWY), 536.5(ACILTV), S537(-ADFGNQTY), 537.5(T), G538(—), V539(AHLMNQST), L540(IM), 540.5(KN), I541(-DEPQRT), 541.5(ACDEMQRV), F542(P), 542.5(QY), G543(APS), 543.5(DHIKLMPQRSTV), K544(-AFST), 544.5(AEFGHIKLMNRVWY), Q545(-ADGHKLNRWY), 545.5(ACEFHILMNPQSTVWY), G546(ACEFHLNPQSTVWY), 546.5(ACDEFGHILMNQSTVWY), S547(-CDFGHLNQVWY), 547.5(ACDGHILMNPQSTVWY), E548(ADFGLMNPS), 548.5(ACDFGHILNPSTWY), K549(-DEFGHLPQSY), 549.5(ADFGLMNQSTVY), T550(-CQWY), 550.5(AEGIKMNPQRSTV), N551(-ACEFGIKPQRSTV), 551.5(ACDEFILMPQST), V552(CQST), 552.5(VW), D553(AEGST), I554(ACHQSTW), E555(AGHNSTVY), K556(CDMQ), 556.5(Q), V557(IL), M558(HIV), I559(FQV), T560(V), D561(INRVY), E562(D), E563(R), I565(T), 565.5(Y), R566(DFHLQY), T567(P), 567.5(E), T568(P), 569.5(M), P570(QS), 570.5(M), V571(ADEFHILMQSTY), A572(CGY), 572.5(WY), T573(ACDGHINSVWY), 573.5(T), E574(ACDFLMSW), 574.5(V), Q575(-CFHILMSTV), 575.5(AWY), Y576(ACFGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(ACFHLNVY), 578.5(ACDEFGHILNSTVY), V579(DFGILMNQSWY), 579.5(ACDEFGILMNPQSTVWY), S580(-CDEFGHILMNPQTVY), 580.5(ACDEFGIKLMNPQSTVWY), T581(-AFGHLNPQWY), 581.5(ACDEFGILMNPQTVWY), N582(-ACDEFGHIKLMPQRSTVWY), 582.5(DFGHNPSTVW), L583(-ADEFGMPSTWY), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMPQSTVWY), R585(-ACDFGHIKLMNPSTVWY), 585.5(ADEFGHILMNPQSTVWY), G586(-DEHMVY), 586.5(ADEFGHILMNPQSTVY), N587(-DEFHILMPQTVY), 587.5(ADEFGHILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(ADEFGHILMNPQSTVY), Q589(-ADEFGINPRSTVWY), 589.5(ACDEFGHIKLMNPQSTVWY), A590(DEFHKLMNQTWY), 590.5(ACDEFGIKLMPQSTVWY), A591(-FGILNPVWY), 591.5(ACDEFGHILMNPQSTVY), T592(-CDEGHLMNPQVY), 592.5(ACDEFGILMNPQSTVWY), A593(-CFLMNPWY), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(ACDEFGHIMNPQSTWY), 595 5(ACDEFGHILMNQSTVW), N596(ADEFGHIMPQSTWY), 596.5(DENY), T597(EFKPR), 597.5(F), Q598(AEGHNSY), G599(SW), V600(LM), 600.5(F), L601(VWY), P602(CMQ), G603(A), M604(CS), V605(ACFMNT), 605.5(N), 606.5(P), D608(ENW), R609(D), D610(Y), 610.5(F), Y612(R), 613.5(Y), Q614(NT), G615(S), P616(AC), I617(KM), 617.5(1), K620(ACGHMN), I621(K), H623(CDFKLSVW), G626(A), H627(AFILMNQW), F628(L), H629(WY), P630(ACGQT), 630.5(C), S631(N), L633(HMY), M634(C), F637(I), L639(FVY), K640(ANRTVWY), 640.5(N), H641(CIK), Q645(LMT), I646(CMT), L647(IY), 1648(C), N650(CILTV), P652(A), V653(CT), A655(S), 655.5(MV), N656(QW), 657.5(CDHLSVW), S658(FIMN), 658.5(ACEHIKLNQRWY), T659(-CEQR), 659.5(ACDEFGIMNSVY), T660(-CDEHKMPQR), 660.5(ADEFHIKLPQWY), F661(AEILMQSTV), 661.5(ACDEFGIKLMPRSVY), S662(CPRW), 662.5(ACDFHIKLMPQRSTVW), A663(IKLMNPY), 663.5(CDGILMNPRSTVWY), A664(DHKNRWY), 664.5(ACHLMPRTVWY), K665(ACFGHIMNQRW), 665.5(AHILNPQRSTWY), F666(CGSWY), 666.5(IPV), A667(CILMRY), 667.5(QY), S668(A), F669(Y), I670(V), T671(NRS), Q672(CEGLS), S674(G), T675(S), V678(C), S679(CEFGHIKLRY), V680(CL), E681(DLNST), I682(M), E683(H), E685(ALQT), L686(T), Q687(IMVW), K688(HP), N690(R), E697(-DHIM), 697.5(GHNPQW), I698(-DL), 699.5(E), T701(S), 701.5(A), S702(IQV), 702.5(Q), N703(DG), 703.5(AEHLTY), Y704(H), 704.5(ADEILNST), N705(CDEHILPTV), 705.5(ENQY), K706(DEGRST), S707(CDEGHILMNQTVY), 707.5(MY), V708(ACEGHNRS), 708.5(1), N709(CDKLPQR), 709.5(CGPQ), V710(AELMNPQ), 710.5(D), D711(GNP), 711.5(QY), T713(CS), T716(CFIKNQRVWY), N717(AEFHRSY), V719(KNQRY), 720.5(N), S721(CFHIKLMNQRTVWY), E722(DQ), P723(ACDEHNQSTV), R724(FHKSWY), P725(ACIQV), I726(LV), R733(K), N734(V), L735(C) 1.5(AGMV), A2(CS), 2.5(AGIMP), A3(-CDEFILQSY), 0.9-fold 3.5(ACDFILMTVY), D4(ACHNSTVWY), 4.5(AFGHNVY), G5(-ACN), 5.5(CFKM), Y6(FI), 6.5(N), L7(F), D9(AV), W10(ACEGIKLPQRSTY), 10.5(HI), L11(ACFIV), 11.5(K), E12(DFGHILNQSWY), D13(CEGIKLNRVWY), T14(CDFGHINPQRSV), L15(AFHTW), S16(G), E17(ACFINQRSTVY), G18(ADS), I19(AEFGHNQSTV), R20(EFHILQSV), Q21(FGHIKNRSTVY), 21.5(FWY), W22(FIVY), 22.5(FILMWY), W23(CDEFGHKLMNQRSTVY), 23.5(ACDEFGHLNQSTWY), K24(ALNPQV), 24.5(CDEHILNY), L25(AEFGHNQRSTV), 25.5(CEGHIKLNQRSTVWY), K26(DHNY), 26.5(HKNQ), P27(T), 27.5(G), 28.5(AEFGILNPQSTVWY), P29(ACDEFGHILQRSTVW), P30(ADFHQRSTY), 30.5(ADEFGIKLNPQRSTY), P31(DFGHIKLNRSY), 31.5(AFILPVY), P32(AEGHIKLNQRSTY), 32.5(ADEFHIKLNPRSTVWY), K33(-ADFGNPQRSTWY), 33.5(CDEFHIKNPQTVWY), P34(-AEFGHKLNQRTVWY), 34.5(CEFGHKPQRSTVWY), A35(EGHKLNQVY), 35.5(CEFGHINPQTVY), E36(-CFGHKNTVW), 36.5(DFHIKLNRSW), R37(-DEGNSVW), 37.5(CDEFIMPQRSW), H38(-ACDELNRVWY), 38.5(ACDEGIKNPRSV), K39(AEFILMRTVWY), 39.5(CDGHINSWY), D40(-ACGLNY), 40.5(CDGQSTY), D41(ACEHQRS), 41.5(CDFNRSTVW), S42(-DKLQRTVWY), 42.5(ACDGKTWY), R43(ASTVY), 43.5(DPRSV), 45.5(M), V46(I), L47(F), P48(LY), G49(CFLMQWY), Y50(HW), 50.5(N), K51(ACFLMNQVY), Y52(D), 53.5(R), P55(Y), 55.5(EKY), 56.5(K), G58(CES), 58.5(F), L59(ACFGKQRW), 59.5(H), D60(ACEHNQSTY), K61(-ACILQRSV), 61.5(GK), G62(QS), E63(ADFHMNV), V65(CFH), N66(AG), E67(ACDLPQRVW), A68(CILPST), A70(CFLY), A71(FGKLNTVWY), L73(CEHKNVY), E74(ACLQTVW), K77(CDSTWY), 77.5(K), A78(FKNRSY), 78.5(ADFHIQR), Y79(-C), 79.5(CK), D80(AHMQTY), 80.5(F), R81(ACDGLQW), 81.5(FHILW), Q82(-CEFGHIMNRTW), 82.5(DFIKLW), L83(ACFGKNQW), 83.5(EFGHIKLQRSTVWY), D84(HILQSV), 84.5(AENQ), S85(-CEKLPWY), 85.5(AFGHKSY), G86(-ACDEHLSVWY), 86.5(FGQSVW), D87(ACFGHKNRSY), 87.5(ADEFGQSWY), N88(ADHLR), P89(ACDH), 89.5(GL), 90.5(EMY), L91(GQRS), K92(AFGINQRSTV), 92.5(RY), Y93(FGHNW), 93.5(Y), N94(-STW), 94.5(AY), H95(ACDEGLRSY), 95.5(W), A96(GQVW), D97(-G), A98(CDIKNWY), 98.5(CE), E99(CFGNSTY), 99.5(FKN), F100(KY), Q101(ADEPVY), 101.5(E), E102(FILQSY), R103(CDGINY), 103.5(ASV), L104(CINT), 104.5(ADEFGIKNSTVW), K105(EFMQRW), 105.5(CFGLQRSVWY), E106(CFGIKNQSTWY), 106.5(EFHINRTVY), D107(-CFKNQTVWY), 107.5(KNPQSW), T108(-CFINPQY), 108.5(AEFGHIQSTY), S109(ACDEFIKLNY), 109.5(AGHNY), F110(ACEHINPY), G111(CE), 111.5(AW), G112(APT), 112.5(CQ), N113(CDFGHIKLQRTVW), 113.5(FHN), L114(ACEFGHN), G115(-ACDEFMPRSV), 115.5(ADEHLMNVW), R116(EGHLMP), 116.5(ADEGHIKLPQRSTVWY), A117(-CEHKQRWY), 117.5(CDEFKLPQW), V118(-DHIKLPRWY), 118.5(ACEGMP), F119(ACHLQSVWY), 119.5(AHIKLNQRSTVY), Q120(-CFIKLRSTW), 120.5(GIKLPR), A121(-EFGHIKNQTVW), 121.5(FGHQR), K122(-ADEFGNRW), 122.5(ADFHIKLPQSTW), K123(CDEFHQSTVWY), 123.5(AFKNPQT), R124(-CFGHIPTWY), 124.5(CEFHKPRSV), V125(-ACDEFHNPQSWY), 125.5(ACEFGQRSW), L126(-CDEFKQRWY), 126.5(CEGRS), E127(-CDHIMPWY), 127.5(ADGILNSTVY), P128(ACDFILQRSTV), 128.5(AGHKLMRSTW), L129(-CDFGKNRSWY), 129.5(ACDFGQRY), G130(CEFQ), 130.5(CDEHKLQRSTY), L131(DEGHIPQTVY), 131.5(ACDEFHLPRW), V132(-CDRSWY), 132.5(EFHIKLNPRVWY), E133(-ADGHLNST), 133.5(EGIPSW), E134(-ADILPW), 134.5(EHINQRW), P135(CDFHKSTWY), 135.5(CDELNPRSTWY), V136(-DEFGHIKQSTWY), 136.5(ACEGHIKLMNPQRSTVWY), K137(-ACDEHLPRSTWY), 137.5(ACDEFGHIKLNPQRSTVWY), T138(-ADEFGHKNPQRW), 138.5(CDEFGHIKNPQRSTVWY), A139(-CDEFGIKLPQRSTWY), 139.5(ACDEGHLNRVY), P140(-ACDEFGHKRSTVWY), 140.5(ACDEHIKNQRVWY), G141(-CDEFHIKLMNQRSTVWY), 141.5(DEFHIKLQRTVY), K142(-ACDEFGILNPQSY), 142.5(ACDEFGHIKLNPQRSTVWY), K143(-EGIRSVY), 143.5(AEFKLPQRSTWY), R144(AHNPSTVW), 144.5(ACEFGHIKLNPQRSTVWY), P145(-ACDEFGHIKLNQRSTVWY), 145.5(DEGIKMNPQRSTVWY), V146(-ACDEFGKLNQRTWY), 146.5(ACEFHIKLNPQRSVY), E147(-ADFGHKLNQTVY), 147.5(ACDEFGHIKRSVW), H148(-ACEGKRTVW), 148.5(AGHKMNPQRTVW), S149(-ACEGHIMNPQRVW), 149.5(ACGHIKLMNPRSTW), P150(CDFHMNQRSVY), 150.5(CDEKLMNPQRSTVWY), V151(-CDGKRSWY), 151.5(DIRWY), E152(ACFHIKLNPQVW), 152.5(DGHKMNQRY), P153(ISV), 153.5(AP), D154(S), 155.5(D), S156(AGT), S157(AEFGKQRTVW), 157.5(DNQTW), G158(D), 158.5(GP), T159(DEFGHIPQR), 159.5(PY), G160(CY), 160.5(CDFHNSWY), K161(-GHLNQSTVWY), 161.5(ADFGILQRSTVW), A162(DIKLPQW), 162.5(ACELNPQRST), G163(-CDFHKNPRTV), 163 5(DEGHIKLNPRSTVWY), Q164(CDHIMRTVW), 164.5(ADEFGIKNPQRSY), Q165(ACHILNPSVW), 165.5(FIPQTV), P166(-ACFGHIKLNQRSTVY), 166.5(FGHINPSTY), A167(QSV), R168(K), R170(K), L171(1), N172(-ACDFHKLQSVWY), 172.5(F), 173.5(ADEFGHINQWY), G174(ACDEFHQSTWY), 174.5(ACEFHIKNQRSY), Q175(-ACDGHKNRST), 175.5(ACDFGIKLNQRSY), T176(ACDFGRY), 176.5(ADENP), G177(-EQ), 177.5(CEGILPTY), D178(-AEIRSWY), 178.5(DFINPRSW), A179(-CEFIKPSW), 179.5(ACDEFGHKNPQRSTVWY), D180(-ACFHIMNQVY), 180.5(ACDFGHILMQRSV), S181(FKLNRTY), 181.5(ACGHNSTVY), V182(ACINPRS), 182.5(CHLPRS), P183(CHLRS), 183.5(CDEGKLMNQY), D184(ACFGHILNPQRTWY), 184.5(CLMPRVY), P185(CDEFGILNSTVWY), 185.5(CEFILPQTVW), Q186(-FGIKNPSTY), 186.5(CDEIKNQSY), P187(-ADFGILNQRST), 187.5(CEGKNPQRWY), L188(CDEFIMPQSTW), 188.5(ADIKLNQSV), G189(-CEIKMNRV), 189.5(EFGLMQRW), Q190(-ACGLMNRSVW), 190.5(ACDFGHPQRSY), P191(AHIKLMNVWY), 191.5(CFIPQRTVW), P192(-ACDFKLMQRSTWY), 192.5(EGHKLMNSVY), A193(CDGHIKLMNPQY), 193.5(DHIMPRSTVW), A194(CFGHIKMQVW), 194.5(CFKMNPT), P195(DEGHKQRSTVY), 195.5(CDHMPQRST), S196(-DFGKNPQRTW), G197(AS), L198(ACDFGNRVWY), G199(D), 199.5(CFHRW), T200(ACFGINQRS), N201(CDH), T202(KPQRSVWY), M203(ACDEFGHIKLPQRSTVWY), A204(DV), T205(EFIKMRVWY), G206(ACDEFHKQRSY), S207(ACDGHRY), G208(ADEFHKLNQRVWY), A209(DEGHKMNQRSVY), P210(CGHQR), M211(LV), A212(G), D213(ACEFGHIKLNPSTW), N214(ACDEFIKLPSVY), N215(CDHILY), E216(ADH), G217(ACDEFHILMNQSTVY), A218(QST), G220(ACMNST), V221(I), N223(ACDHIMQSTY), S225(CQR), H229(ACDSTV), D231(C), T233(DHIMQSVY), M235(LV), 236.5(D), D237(CGH), 237.5(DEGHKNQ), R238(CHS), 238.5(CFIY), I240(FHV), T242(CFHRSY), S243(A), T244(C), T246(IV), A248(S), L249(CV), P250(AQS), T251(CLPSV), Y252(F), N253(D), N254(CGQS), H255(A), L256(ACGHKMNQRSV), K258(LQ), Q259(AGHKPRS), 260.5(MNQ), S261(ACGNT), S262(T), 262.5(D), Q263(-DEFGHLTWY), S264(-ADEHILMQTVWY), 264.5(D), 265.5(ADEMNQST), A266(S), 266.5(AE), S267(AENPT), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(EFINV), F273(HWY), S276(AK), G280(A), Y281(FH), F282(LMVWY), F284(CY), F287(Y), H288(N), C289(S), S292(C), D295(AE), N303(TV), F306(W), R307(-CDIKMSTY), 307.5(FKN), P308(-NV), K309(ACQS), R310(ACFGIKLNSTVW), L311(CFIY), N312(CDHLMQTWY), F313(V), K314(CFGHLMNQRSTVWY), L315(V), I318(FL), 323.5(CT), T324(AHLMN), 324.5(CFKMRT), Q325(CHKRV), 325.5(ACFGHIKRST), N326(CDGLMRTWY), 326.5(N), D327(AGQT), 327.5(AGHT), G328(AN), 328.5(G), T329(ACFIY), T330(KNQS), T331(FHNY), I332(ACSV), 332.5(K), A333(CDFHNSTY), N334(QS), L336(I), T339(C), V342(ACIM), F343(M), T344(CDEFIKLMNRSV), S346(ACEFHIKLMPQVWY), Y348(AEFGHNRSW), Q349(AGMS), V353(CIT), L354(AIMQTVY), G355(S), S356(A), H358(ACGLNQST), Q359(DEHKMTV), C361(A), L362(I), F365(W), A367(CP), V369(CIPT), F370(Y), M371(ACIKLNQSTV), V372(CI), Y375(FW), G376(CS), Y377(F), L378(ACV), T379(ACGSV), 379.5(DE), L380(CEMNQTVY), 380.5(E), N381(ACDGMQS), N382(CDEST), 382.5(D), 383.5(E), S384(AG), 384.5(D), Q385(ACDEIKLMRSTVY), 385.5(ADEHKLPT), A386(-EGHLNPRSTVWY), 386.5(CDEPY), V387(CDEIQTWY), 387.5(EGKP), G388(EIM), R389(GIL), S390(ADGNT), 390.5(C), S391(AG), Y393(F), C394(V), 394.5(N), 395.5(W), Y397(AGN), P399(EIMQRS), Q401(ACDEFIKLMNRSVWY), L403(C), R404(K), 404.5(N), T405(SY), G406(DENQ), 406.5(Q), 407.5(H), N408(ADFGHLMQSTWY), 408.5(CMN), F409(CHW), T410(CEFGHLMQVY), F411(ACLTVW), S412(EFGIKMNQTVWY), 412.5(Q), Y413(FM), T414(EFGIKLMNQ), 414.5(D), 415.5(ADGNPQS), E416(ADG), 416.5(CEGKMNRS), D417(AEGIKMNSTV), 417.5(DEKLMQRS), V418(CGHLMQS), P419(DEGIKLNRSTVW), F420(LM), S422(A), S423(CLT), Y424(FHT), A425(-CFQSV), H426(N), S427(ANT), Q428(EHNS), S429(CGHPY), L430(MV), D431(M), 431.5(Y), R432(-I), L433(IMSV), 433.5(F), M434(A), L437(ANSV), I438(GHMT), D439(KNPSTY), 441.5(M), L442(ITV), 444.5(CLM), L445(CFIMVY), 445.5(ACDEGHILMNQSTVY), S446(-CDEGHILMNQTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-ACDEFGHILMNPQSTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQSVWY), 448.5(ACDEFGHILMNPQSY), N449(-ACDEFGHILMPTVWY), 449.5(AEGHLMNPST), T450(-ACDFGLMNPSY), 450.5(ADEFGHLPQSTV), P451(-IL), 451.5(GNP), S452(-DEFGILNPQIY), 452.5(ALV), G453(-AEFHIKLMNPQST), 453.5(FILMNY), T454(-ADEFGHILMNPQSV), 454.5(ADEFGLMPY), T455(-ADEILMNPQSVY), 455.5(FHNPQ), T456(ADEFGHIKLMNPQSY), 456.5(DEGHILPS), Q457(EFGHILMPS), 457.5(ADEFGHILMNPTWY), S458(-DEFHMNVW), 458.5(CDEGHILMNQSTVW), R459(-DEGINPY), 459.5(DEP), L460(AHIMPTWY), 460.5(W), Q461(DEFHIKMSTV), F462(Y), 462.5(N), S463(ACDGKMNQRTV), 463.5(F), Q464(CIKLMRTV), 464.5(SY), A465(G), G466(AD), 466.5(E), A467(EKMS), S468(ACDEFHLNPQVWY), D469(V), 469.5(E), R471(DEGQSTV), D472(GHLMQSTVWY), Q473(FHSY), S474(ACIY), R475(HK), 475.5(SW), N476(CSTW), 476.5(L), W477(FY), 477.5(L), L478(FI), 478.5(L), P479(GIT), 479.5(D), G480(Y), P481(ACV), 481.5(GHY), C482(AFGHNQSWY), 482.5(F), Y483(ACHITVW), 483.5(FHQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACFGILMNSTY), 485.5(EIKMQY), Q486(ACDEFGILMPSVWY), 486.5(FV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(ACEFGHILMNQTWY), 488.5(ACDEFGILMPQSVWY), S489(-ACEFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACDEFGHILMPQRSTVWY), T491(-AGMPQSY), 491.5(ADEFGHIKLMNPQSTVWY), S492(-CDFGHLMPQTV), 492.5(ADEGHILMPQSTVY), A493(-HLQRSTV), 493.5(ADEFGHIKLMNPQSTVWY), D494(EFR), 494.5(ADEFGHILMNPQSTVWY), N495(-ADEFGHILMPQSTVWY), 495 5(ACDEFGILMNPQSTVWY), N496(-ACDEFGILMPQTVWY), 496.5(ADEFGHILMNPQSTVWY), N497(-CEFGHLMSTVWY), 497.5(ADEFGILMNPQSTVY), S498(-CDEGINPQRTVW), 498.5(ADEFGHILMNPQSTVWY), E499(CIPS), 499.5(ACDEGHILMNPQRSTVWY), Y500(-ACDEGHIKLMNPQSTVW), 500.5(ADEFGHILMNPQSTVWY), S501(-ACDEFGHILMNPRTVWY), 501.5(ACDEFGHILMNPQRSTVWY), W502(-ACDEFGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGHILMNPQSWY), 503.5(ADEFGILMNPQSTVW), G504(-ACDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-FGILMNPQS), 506.5(ACIPSTVW), K507(-ACEFHILMNVWY), Y508(-DFIMSTV), H509(EFKNQR), 509.5(K), L510(I), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(HIV), D514(ACEFGILMQSTV), S515(ACINTV), L516(ACIMRS), V517(CL), 517.5(C), N518(AILQRTVY), P521(CFMNS), 521.5(M), A522(CSV), M523(CLV), 523.5(A), A524(GPS), 524.5(ACGHSTW), S525(CMNPT), 525.5(ACDEFGHILMNPQSTVWY), H526(-DEFGIKLMPRVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILPRSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AEFMPQSVW), 528.5(DEPT), D529(ES), 529.5(ADEGLMNPQTV), E530(DNPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGHILMNPQSTVY), 531.5(ADEFGIKLMNPQRSTVY), K532(-ACDEGPR), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHILNQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-ACDEFGHILMNPSTVWY), 536.5(ACILTV), S537(-ACDFGHNQTY), 537.5(T), G538(—), V539(ACHILMNQST), L540(IM), 540.5(KN), I541(-ADEFGKPQRTV), 541.5(ACDEGMQRSTV), F542(GP), 542.5(QY), G543(APS), 543.5(ADHIKLMPQRSTV), K544(-AFGINQSTVY), 544.5(ADEFGHIKLMNRSTVWY), Q545(-ADFGHKLNPRSTWY), 545 5(ACEFGHILMNPQSTVWY), G546(ACEFHLNPQSTVWY), 546.5(ACDEFGHILMNQSTVWY), S547(-CDFGHILNQVWY), 547.5(ACDGHILMNPQSTVWY), E548(ADFGILMNPSWY), 548.5(ACDFGHILNPSTWY), K549(-DEFGHILNPQSTY), 549.5(ADEFGLMNQSTVY), T550(-CQSWY), 550.5(ADEGIKMNPQRSTV), N551(-ACDEFGHIKMPQRSTVY), 551.5(ACDEFILMPQST), V552(CEILQST), 552.5(VW), D553(AEGST), I554(ACEHQSTVW), E555(AGHNSTVY), 555.5(N), K556(CDMQW), 556.5(Q), V557(IL), M558(AHILNSTV), I559(FQV), T560(AV), D561(EHILNRSVY), E562(AD), E563(CR), I565(LT), 565.5(Y), R566(DFHLMQY), T567(P), 567.5(E), T568(P), 569.5(M), P570(QS), 570.5(M), V571(ACDEFHILMQSTY), A572(CGY), 572.5(WY), T573(ACDGHINSVWY), 573.5(T), E574(ACDFLMQSW), 574.5(V), Q575(-CFGHIKLMNSTVW), 575.5(AWY), Y576(ACFGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(ACFHLNQVY), 578.5(ACDEFGHILNSTVY), V579(CDEFGHILMNQSTWY), 579.5(ACDEFGILMNPQSTVWY), S580(-CDEFGHILMNPQTVWY), 580.5(ACDEFGIKLMNPQRSTVWY), T581(-AFGHLNPQSWY), 581.5(ACDEFGILMNPQTVWY), N582(-ACDEFGHIKLMPQRSTVWY), 582.5(DFGHNPSTVW), L583(-ADEFGMPSTVWY), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMNPQSTVWY), R585(-ACDFGHIKLMNPSTVWY), 585.5(ADEFGHILMNPQSTVWY), G586(-DEFHLMTVY), 586.5(ACDEFGHILMNPQSTVY), N587(-DEFHILMPQSTVY), 587.5(ADEFGHILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(ADEFGHILMNPQSTVY), Q589(-ADEFGIMNPRSTVWY), 589.5(ACDEFGHIKLMNPQSTVWY), A590(DEFHKLMNQTWY), 590.5(ACDEFGIKLMNPQSTVWY), A591(-FGILNPVWY), 591.5(ACDEFGHILMNPQSTVY), T592(-CDEFGHLMNPQVY), 592.5(ACDEFGILMNPQSTVWY), A593(-CFLMNPWY), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(ACDEFGHILMNPQSTWY), 595.5(ACDEFGHILMNQSTVW), N596(ADEFGHIMPQSTWY), 596.5(DENY), T597(EFGKPRV), 597.5(F), Q598(AEFGHNSY), G599(SW), V600(LM), 600.5(F), L601(MVWY), P602(CMQ), G603(A), M604(ACQS), V605(ACFMNT), 605.5(N), 606.5(P), D608(ENQW), R609(D), D610(Y), 610.5(F), Y612(R), 613.5(Y), Q614(NT), G615(S), P616(ACH), I617(KM), 617.5(1), K620(ACGHMNQS), I621(HKLQRTV), H623(CDFKLSVW), G626(A), H627(AFGILMNQVW), F628(ILM), H629(WY), P630(ACGQTV), 630.5(CV), S631(AHNT), L633(FHMY), M634(CI), F637(IW), L639(FVY), K640(AFNRTVWY), 640.5(N), H641(CIKL), P644(W), Q645(LMT), I646(CLMTV), L647(FIY), I648(CV), N650(CILTV), P652(A), V653(CT), A655(S), 655.5(LMV), N656(FQTW), 657.5(CDFGHLMNQSTVW), S658(AFGIMNQT), 658.5(ACEFGHIKLNQRWY), T659(-CEGIQRY), 659.5(ACDEFGIMNPSVY), T660(-CDEGHKMPQRV), 660.5(ADEFGHIKLPQRWY), F661(AEILMQSTVY), 661.5(ACDEFGIKLMNPRSVY), S662(CPQRW), 662.5(ACDEFGHIKLMNPQRSTVW), A663(FHIKLMNPRY), 663.5(CDGHILMNPRSTVWY), A664(CDHKNRWY), 664.5(ACDEHLMNPRTVWY), K665(ACFGHIMNPQRSTW), 665.5(AHILNPQRSTVWY), F666(CGLNSWY), 666.5(IPV), A667(CEGILMNPRY), 667.5(HNQY), S668(AG), F669(Y), I670(V), T671(NRS), Q672(CEGLS), S674(G), T675(S), V678(ACT), S679(CEFGHIKLRY), V680(CL), E681(DLMNSTV), I682(M), E683(HIQ), E685(ADLQT), L686(T), Q687(IMVW), K688(HP), N690(R), E697(-DHIMPR), 697.5(GHNPQW), I698(-DL), 699.5(E), T701(S), 701.5(A), S702(IQV), 702.5(Q), N703(DG), 703.5(AEHLTY), Y704(FH), 704.5(ADEILNST), N705(CDEFHIKLMPQTV), 705.5(ENQY), K706(DEGRST), S707(CDEFGHILMNQTVY), 707.5(MY), V708(ACEGHNRST), 708.5(1), N709(CDKLPQRV), 709.5(CGPQ), V710(AELMNPQ), 710.5(D), D711(GNP), 711.5(QY), T713(ACS), T716(ACFIKLNQRVWY), N717(ADEFHRSY), V719(FKLNQRY), 720.5(N), S721(CFHIKLMNQRTVWY), E722(DQ), P723(ACDEHNQSTV), R724(FHKNSWY), P725(ACILQV), I726(LV), R733(K), N734(AEISV), L735(C)

TABLE 8 Amino acid alterations that reduce delivery to spleen. Fold increase of Amino acid alteration efficiency D439(K), 444.5(CLM), 445.5(ACELMNST), 446.5(CN), 447.5(INP), 0.1-fold 460.5(W), G466(Q), 476.5(L), 478.5(L), R484(EFGLMNSTVY), 485.5(IKM), Q486(IVW), R487(CDFGILNSTVW), 487.5(F), 488.5(WY), K490(EP), 493.5(K), 497.5(D), S498(D), 498.5(DE), E499(C), 499.5(CDE), 503.5(IMQW), 504.5(FLNSTY), A505(E), 505.5(ACLQTVW), 506.5(ACISV), 523.5(A), 524.5(GHS), 525.5(ACDFGHILNQSTVWY), H526(EL), 526.5(CDEFHILMNPQSTWY), K527(-W), 527.5(ACDEHILMPSVWY), 528.5(D), 529.5(D), 530.5(IW), 531.5(IV), K532(P), 532.5(CGHIPQSTVWY), 533.5(CFNPQW), 534.5(F), P535(-F), Q536(—), 536.5(CITV), V571(H), 575.5(W), 576.5(W), G577(D), 577.5(DI), 578.5(CEITY), 579.5(Q), S580(—), T581(DE), 581.5(CY), N582(F), Q584(W), 586.5(F), R588(N), 590.5(K), A591(E), 591.5(DEP), T592(DE), 592.5(CMP), A593(-P), 593.5(FGPW), 595.5(CD), 596.5(DEY), 605.5(N) D180(M), 266.5(E), 268.5(E), 379.5(E), 380.5(E), 382.5(D), 433.5(F), 0.2-fold D439(K), 444.5(CLM), L445(F), 445.5(ACEGHILMNQSTV), S446(—), 446.5(ACEFHILMNPQSTVWY), R447(EFIWY), 447.5(DEGHILMNPQVW), T448(-DEHINPQV), 448.5(DEP), N449(P), G453(K), 453.5(Y), 454.5(F), S458(E), R459(D), L460(Y), 460.5(W), 462.5(N), 463.5(F), G466(Q), 469.5(E), R471(EG), S474(I), 476.5(L), 478.5(L), P479(T), 479.5(D), C482(W), 483.5(V), R484(EFGHILMNQSTVY), 485.5(EIKM), Q486(EFIMVWY), 486.5(FV), R487(ACDEFGHILMNPQSTVWY), 487.5(FW), V488(H), 488.5(DEILPWY), S489(DLMPQWY), 489.5(DPTW), K490(-CDEPW), 490.5(DE), T491(DP), 491.5(DEFKW), 492.5(DE), A493(D), 493.5(DEK), D494(F), 494.5(DE), N495(FLP), 495.5(CI), N496(D), N497(—), 497.5(DE), S498(DE), 498.5(DE), E499(C), 499.5(ACDEGPQ), Y500(ACDEIPV), 500.5(DELQ), 501.5(CEGNW), W502(-EP), 502.5(ADEFGMNPTWY), T503(DE), 503.5(DEFGILMNQSTVW), G504(-DILNPTVY), 504.5(ACDEFGILMNPQ STVWY), A505(DEHILMNQY), 505.5(ACDEGILMPQSTVW), T506(-F), 506.5(ACIPSTV), K507(-ACEINSWY), Y508(—), H509(FN), 509.5(K), 510.5(K), 517.5(C), 523.5(A), 524.5(ACGHST), 525.5(ACDEFGHILMNPQSTVWY), H526(DEILPV), 526.5(ACDEFGHILMNPQSTVWY), K527(-FHLPWY), 527.5(ACDEFGHILMPQSTVWY), 528.5(DET), 529.5(ADELMPV), E530(PW), 530.5(CDEFILMNQTVWY), 531.5(ADEFGILNPQTV), K532(-ACFGHILMNPQSTVW), 532.5(ACGHILMPQSTVWY), F533(ADEGSTV), 533.5(ACEFGHILNPQSTVWY), 534.5(F), P535(-F), Q536(-DP), 536.5(ACILTV), S537(—), 537.5(T), 559.5(A), V571(EHS), 571.5(G), 573.5(T), Q575(K), 575.5(WY), Y576(V), 576.5(W), G577(D), 577.5(ACDEHIMQS), S578(DE), 578.5(ACEFILSTY), V579(DEW), 579.5(DEFILNQTY), S580(-DEQ), T581(DEP), 581.5(CDIQY), N582(-CFILY), 582.5(FGT), 583.5(PTVY), Q584(GVW), 584.5(DEIMPV), R585(AEFGHILMNQSTVWY), 585.5(AFILMPSTVY), G586(-DE), 586.5(ACFGILMNPQSTV), N587(-DE), 587.5(ADILMNTV), R588(ACFHILMNQSVWY), 588.5(GIV), 589.5(CFHKPV), A590(M), 590.5(CGKPQST), A591(DEW), 59E5(CDEFGILMPTV), T592(-DE), 592.5(ACEFGILMNPQSTVWY), A593(-DEP), 593.5(ACDFGILMNPSTVWY), D594(LT), 594.5(ACFGHILMPQTVWY), 595.5(CDEFGIQTVW), N596(DE), 596.5(DEY), 605.5(N), 606.5(P), R609(D), D610(Y), 617.5(1) D180(M), 180.5(M), S261(D), 264.5(D), 266.5(E), S267(E), 267.5(E), 0.3-fold N268(E), 268.5(E), H271(E), 379.5(DE), L380(E), 380.5(E), 382.5(D), 383.5(E), 386.5(D), 431.5(Y), 433.5(F), D439(K), 444.5(CLM), L445(FY), 445.5(ACDEGHILMNQSTV), S446(-EL), 446.5(ACDEFGHILMNPQSTVWY), R447(-DEFIPVWY), 447.5(CDEGHILMNPQVW), T448(-DEGHILNPQVW), 448.5(DEP), N449(DEP), 449.5(E), G453(IK), 453.5(Y), 454.5(EF), T455(E), 457.5(D), S458(DE), 458.5(DE), R459(DE), L460(WY), 460.5(W), Q461(D), 462.5(N), 463.5(F), 464.5(Y), G466(Q), 469.5(E), R471(EGSTV), S474(I), 475.5(W), 476.5(L), 478.5(L), P479(GT), 479.5(D), G480(Y), C482(W), 482.5(F), 483.5(QV), R484(EFGHILMNQSTVY), 485.5(EIKMQY), Q486(DEFIMPVWY), 486.5(FV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(EFGHY), 488.5(DEILPSWY), S489(-DEFGKLMPQTVWY), 489.5(ADEGPQRSTW), K490(-CDEFGLNPWY), 490.5(CDEFIPVY), T491(DEP), 491.5(DEFHIKLMWY), 492.5(DEY), A493(DE), 493.5(DEHKP), D494(FR), 494.5(DEGP), N495(-AEFGILMPQSTVWY), 495.5(CDEGILPTV), N496(-CDEGILM), 496.5(DE), N497(-CEH), 497.5(DE), S498(-CDEFPR), 498.5(DE), E499(CIP), 499.5(ACDEGLMNPQSTVW), Y500(ACDEGIKLPV), 500.5(ADEFGILNPQST), S501(D), 501.5(ACDEFGHILMNQSTVWY), W502(-ACDEMP), 502.5(ADEFGILMNPSTVWY), T503(-DE), 503.5(ADEFGILMNPQSTVW), G504(-CDEFILNPSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-DEFHILMNPQWY), 505.5(ACDEFGILMPQSTVWY), T506(-FLM), 506.5(ACIPSTVW), K507(-ACEFHILMNQSTVWY), Y508(—), H509(CEFN), 509.5(K), 510.5(K), R513(C), D514(FILV), 517.5(C), 521.5(MS), 523.5(A), 524.5(ACGHST), S525(HM), 525.5(ACDEFGHILMNPQSTVWY), H526(DEILMPV), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFHILPQVWY), 527.5(ACDEFGHILMPQSTVWY), 528.5(DET), 529.5(ADEGLMNPQV), E530(PW), 530.5(ACDEFGILMNQSTVWY), 531.5(ADEFGILMNPQRSTVY), K532(-ACFGHILMNPQSTVWY), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGIQSTV), 533.5(ACDEFGHILNPQSTVWY), 534.5(F), P535(-F), Q536(-DPW), 536.5(ACILTV), S537(—), 537.5(T), G538(—), 559.5(A), R566(D), T568(P), P570(Q), 570.5(M), V571(ADEFHS), 571.5(G), 572.5(Y), T573(D), 573.5(MT), Q575(DK), 575.5(FWY), Y576(HMV), 576.5(EFWY), G577(D), 577.5(ACDEHIMQSTV), S578(DE), 578.5(ACDEFILSTVY), V579(DEW), 579.5(DEFGILMNQTVY), S580(-DEILMQY), 580.5(DEN), T581(-DEP), 581.5(ACDEILMPQY), N582(-ACEFHILMPTVWY), 582.5(DFGHPTVW), L583(-P), 583.5(GNPQSTVY), Q584(DFGILNPTVWY), 584.5(ACDEFGHILMNPQSTV), R585(-ACEFGHILMNQSTVWY), 585.5(ADFGILMNPQSTVWY), G586(-DE), 586.5(ACDFGHILMNPQSTV), N587(-DEILQVY), 587.5(ADEFGILMNPQSTV), R588(-ACEFHILMNPQSTVWY), 588.5(DEGILMPV), Q589(-DR), 589.5(ACDEFHKNPTVW), A590(DELMNQ), 590.5(ACEGIKMPQSTW), A591(-DEWY), 591.5(ACDEFGHILMNPQSTV), T592(-DEP), 592.5(ACDEFGILMNPQSTVWY), A593(-DEP), 593.5(ACDEFGILMNPQSTVWY), D594(LT), 594.5(ACDFGHILMPQSTVWY), V595(DEGHW), 595.5(ACDEFGILQTVW), N596(DEI), 596.5(DEY), T597(E), Q598(FY), 605.5(N), 606.5(P), 607.5(P), D608(W), R609(D), D610(Y), 617.5(I), 640.5(N), 667.5(Y) 4.5(H), 45.5(M), 50.5(N), G58(C), S156(T), D180(M), 180.5(M), 183.5(M), 0.5-fold L256(A), S261(D), S262(T), 262.5(E), Q263(G), S264(W), 264.5(D), 265.5(D), 266.5(E), S267(E), 267.5(E), N268(DE), 268.5(E), 269.5(D), H271(E), Q325(K), 379.5(DE), L380(E), 380.5(E), N381(D), N382(DE), 382.5(D), 383.5(E), 384.5(D), 386.5(D), V387(E), G406(E), A425(—), Q428(E), 431.5(Y), R432(I), 433.5(F), D439(K), 441.5(M), 444.5(CLM), L445(FY), 445.5(ACDEGHILMNQSTVY), S446(-DEILQV), 446.5(ACDEFGHILMNPQSTVWY), R447(-DEFILMPTVWY), 447.5(ACDEFGHILMNPQVWY), T448(-ACDEFGHILMNPQVWY), 448.5(CDEFNPWY), N449(DEFMPW), 449.5(E), T450(E), 450.5(F), 451.5(N), 452.5(AL), G453(DEFIKLNS), 453.5(FILMNVY), T454(-FI), 454.5(DEFLM), T455(-DEMY), 455.5(P), T456(-DEK), 456.5(DE), Q457(DEP), 457.5(DEPW), S458(DEFMWY), 458.5(DEFILMNTWY), R459(-ACDEGIPTV), 459.5(DE), L460(FHWY), 460.5(W), Q461(DE), 462.5(N), S463(G), 463.5(F), 464.5(SY), G466(Q), 469.5(DE), R471(ACDEGMQSTV), D472(Y), S474(I), R475(H), 475.5(SW), N476(TW), 476.5(L), 477.5(L), 478.5(L), P479(GT), 479.5(D), G480(Y), 481.5(H), C482(W), 482.5(FM), Y483(TVW), 483.5(FQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACGLST), 485.5(EIKMQY), Q486(ADEFGILMNPVWY), 486.5(FV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(EFGHWY), 488.5(ACDEGILMPQSWY), S489(-CDEFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGHILMNPQSTVWY), 490.5(ACDEFHIMPQRTVWY), T491(-DEP), 491.5(ACDEFHIKLMNPQSTVWY), S492(-E), 492.5(ADEGHILSVY), A493(-DEHR), 493.5(ADEFHIKLMPQSVWY), D494(FR), 494.5(DEGILMPTVY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMPQSTVW), N496(-ACDEGILMPQVW), 496.5(DEFQW), N497(-CEHW), 497.5(DE), S498(-CDEFILPRVW), 498.5(DEFLPQY), E499(CIPQS), 499.5(ACDEGHILMNPQRSTVWY), Y500(-ACDEGIKLNPQV), 500.5(ADEFGHILMNPQSTVY), S501(-DILNVW), 501.5(ACDEFGHILMNQRSTVWY), W502(-ACDEGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-DEGLMPW), 503.5(ADEFGILMNPQSTVW), G504(-CDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-DEFHILMNPQTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-FGILMV), 506.5(ACIPSTVW), K507(-ACEFHILMNQSTVWY), Y508(—), H509(CEFNQ), 509.5(K), 510.5(K), N511(EWY), 512.5(S), R513(CIV), D514(ACEFILMQV), 517.5(C), 521.5(MS), M523(V), 523.5(A), 524.5(ACGHSTW), S525(HM), 525.5(ACDEFGHILMNPQSTVWY), H526(-ADEGILMPQTVW), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFHILMPQSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AFQ), 528.5(DEPT), D529(S), 529.5(ADEGLMNPQTV), E530(NPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACGHILMNV), 531.5(ADEFGIKLMNPQRSTVY), K532(-ACDEFGHILMNPQSTVWY), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGILMNQSTV), 533.5(ACDEFGHILNPQSTVWY), F534(LVY), 534.5(F), P535(-AF), Q536(-ADEGPWY), 536.5(ACILTV), S537(-DY), 537.5(T), G538(—), V539(H), 542.5(Y), D553(T), 1554(E), 559.5(A), E562(A), R566(DEFY), 567.5(E), T568(P), P570(Q), 570.5(M), V571(ADEFHMQSY), 571.5(G), A572(Y), 572.5(WY), T573(DGV), 573.5(MT), E574(D), 574.5(V), Q575(DGKL), 575.5(AFWY), Y576(ACGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(DEFILNWY), 578.5(ACDEFGHILNSTVY), V579(ADEFGSWY), 579.5(ACDEFGILMNPQTVWY), S580(-DEFHILMNPQY), 580.5(ACDEFGKLMNPRVW), T581(-DEKP), 581.5(ACDEGILMNPQVWY), N582(-ACDEFHILMPQRSTVWY), 582.5(DFGHPSTVW), L583(-DGP), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILNPSTVWY), 584.5(ACDEFGHILMNPQSTVWY), R585(-ACDEFGHIKLMNPQSTVWY), 585.5(ADEFGHILMNPQSTVWY), G586(-DEFMW), 586.5(ACDEFGHILMNPQSTV), N587(-DEGHILMPQVY), 587.5(ADEFGILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(DEFGHILMPTVY), Q589(-DEPR), 589.5(ACDEFGHIKNPSTVWY), A590(DEHKLMNQY), 590.5(ACDEFGIKLMNPQSTVWY), A591(-DEFPWY), 591.5(ACDEFGHILMNPQSTVWY), T592(-DEGPV), 592.5(ACDEFGILMNPQSTVWY), A593(-DEP), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(DEGHLMNPQWY), 595.5(ACDEFGHILMNQSTVW), N596(DEIP), 596.5(DENY), T597(DE), 597.5(F), Q598(FHY), G599(W), 600.5(F), L601(Y), 605.5(N), 606.5(P), 607.5(P), D608(W), R609(D), D610(Y), 610.5(F), 613.5(Y), I617(K), 617.5(1), 640.5(N), T660(H), 662.5(F), 667.5(Y), K706(DEG) 4.5(H), 38.5(G), K39(E), 45.5(M), G49(H), Y50(H), 50.5(N), G58(C), 0.7-fold E74(I), S156(T), 157.5(P), D180(M), 180.5(M), 183.5(M), 184.5(M), 189.5(M), P192(M), A194(C), R238(H), H255(N), L256(ACGHS), 260.5(DST), S261(D), 261.5(S), S262(T), 262.5(EGINS), Q263(AGW), S264(DEW), 264.5(D), 265.5(DE), 266.5(E), S267(E), 267.5(E), N268(DEQ), 268.5(E), 269.5(D), N270(CDGY), H271(E), D295(E), L311(1), 324.5(K), Q325(KR), N326(L), T330(K), Y348(D), T379(A), 379.5(DE), L380(EY), 380.5(E), N381(DGQ), N382(DE), 382.5(D), 383.5(E), 384.5(D), Q385(T), A386(E), 386.5(DE), V387(DE), 387.5(EGP), R389(I), S390(DN), P399(M), 404.5(N), G406(DE), 408.5(N), F409(CH), S412(W), 416.5(N), 417.5(M), A425(-Q), H426(N), Q428(EH), S429(Y), D431(M), 431.5(Y), R432(-I), L433(D), 433.5(F), I438(K), D439(HKT), 441.5(M), L442(FI), 444.5(CLM), L445(CFMY), 445.5(ACDEGHILMNQSTVY), S446(-DEHILMNQTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-ACDEFGHILMNPQSTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQVWY), 448.5(ACDEFGILMNPWY), N449(-DEFILMPVW), 449.5(AEGHLNPS), T450(-CDEPW), 450.5(DEFHV), 451.5(N), S452(-DEFPVW), 452.5(AILPV), G453(-ADEFHIKLMNPQSTV), 453.5(FILMNTVY), T454(-DEFILMP), 454.5(DEFLMP), T455(-DEILMQY), 455.5(FLPQY), T456(-DEGHIKP), 456.5(ADEGHILNP), Q457(DEGLP), 457.5(ADEGHILPWY), S458(DEFHMNVWY), 458.5(DEFGHILMNTWY), R459(-ACDEGILMNPQSTVWY), 459.5(DEP), L460(AFHMPWY), 460.5(W), Q461(DEIKV), F462(Y), 462.5(N), S463(CDGKNQ), 463.5(F), Q464(V), 464.5(SY), G466(Q), A467(HMY), S468(CDF), 469.5(DE), R471(ACDEGMQSTV), D472(Y), Q473(FHY), S474(I), R475(H), 475.5(SW), N476(TW), 476.5(L), W477(Y), 477.5(L), L478(FI), 478.5(L), P479(GT), 479.5(D), G480(Y), 481.5(HY), C482(FGWY), 482.5(FM), Y483(ACFHITVW), 483.5(FQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACFGILNSTVY), 485.5(EIKMQY), Q486(ACDEFGILMNPSVWY), 486.5(FV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(AEFGHNQTWY), 488.5(ACDEFGILMPQSVWY), S489(-ACDEFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACDEFGHILMNPQRSTVWY), T491(-DEP), 491.5(ACDEFGHIKLMNPQSTVWY), S492(-CEF), 492.5(ADEGHILMPQSTVY), A493(-DEFHILRWY), 493.5(ADEFGHIKLMNPQSTVWY), D494(FR), 494.5(ADEGHILMPSTVWY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMPQSTVWY), N496(-ACDEFGILMPQTVW), 496.5(ADEFGHILPQWY), N497(-CEFHIMSVWY), 497.5(ADEGMPY), S498(-CDEFILMPRVWY), 498.5(DEFGILMPQSY), E499(CIPQS), 499.5(ACDEGHILMNPQRSTVWY), Y500(-ACDEGIKLMNPQSTV), 500.5(ADEFGHILMNPQSTVWY), S501(-CDEFILNTVW), 501.5(ACDEFGHILMNQRSTVWY), W502(-ACDEFGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGILMPQVWY), 503.5(ADEFGILMNPQSTVW), G504(-ACDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-FGILMNPQV), 506.5(ACIPSTVW), K507(-ACEFHILMNQSTVWY), Y508(-DS), H509(CEFNQ), 509.5(K), L510(I), 510.5(K), N511(DELMQWY), 512.5(S), R513(CHIV), D514(ACEFGILMQTV), L516(ACST), V517(L), 517.5(C), N518(R), P521(MS), 521.5(MS), M523(CV), 523.5(A), A524(GP), 524.5(ACGHSTW), S525(AGHMNP), 525.5(ACDEFGHILMNPQSTVWY), H526(-ACDEFGIKLMNPQSTVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILMPQSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AFMPQSVW), 528.5(DEPT), D529(S), 529.5(ADEGLMNPQTV), E530(NPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGHILMNPQSTVY), 531.5(ADEFGIKLMNPQRSTVY), K532(-ACDEFGHILMNPQRSTVWY), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHILMNQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-ACDEFGNPTWY), 536.5(ACILTV), S537(-ADFGQY), 537.5(T), G538(—), V539(HLM), 540.5(K), 541.5(Q), F542(P), 542.5(Y), 543.5(CQRY), K544(Q), 544.5(AIKRVY), 545.5(CFNPQVWY), G546(Y), 546.5(EGSTWY), S547(-Q), 547.5(ADNVY), E548(FN), 548.5(CDHMPY), K549(-DILW), 549.5(DH), T550(C), 550.5(KR), N551(CDEIRTV), 551.5(CDEM), D553(AT), I554(EHQ), E555(GHTV), K556(EMW), M558(A), 558.5(E), 559.5(A), T560(A), 560.5(A), E562(A), I565(T), R566(ACDEFGHILMNTVWY), 567.5(E), T568(P), 569.5(M), P570(QS), 570.5(M), V571(ADEFHLMQSTY), 571.5(G), A572(CY), 572.5(VWY), T573(ACDGINSVW), 5 73.5(MT), E574(DQ), 574.5(V), Q575(-CDGIKLMSTVY), 575.5(AFWY), Y576(ACGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(DEFGILNWY), 578.5(ACDEFGHILNSTVY), V579(ACDEFGHLMNQSTWY), 579.5(ACDEFGILMNPQSTVWY), S580(-DEFHILMNPQVY), 580.5(ACDEFGKLMNPQRSTVWY), T581(-CDEFGKPWY), 581 5(ACDEFGILMNPQTVWY), N582(-ACDEFGHIKLMPQRSTVWY), 582.5(DFGHNPSTVW), L583(-ADEGPST), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMNPQSTVWY), R585(-ACDEFGHIKLMNPQSTVWY), 585.5(ADEFGHILMNPQ STVWY), G586(-DEFHMNWY), 586.5(ACDEFGHILMNPQSTVY), N587(-DEFGHILMPQSTVY), 587.5(ADEFGILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(ADEFGHILMPQSTVY), Q589(-DEIPRW), 589.5(ACDEFGHIKLNPQSTVWY), A590(DEFHKLMNQWY), 590.5(ACDEFGIKLMNPQSTVWY), A591(-CDEFIMNPTVWY), 591.5(ACDEFGHILMNPQSTVWY), T592(-ADEFGHMNPQVY), 592.5(ACDEFGILMNPQSTVWY), A593(-DEP), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(ADEFGHLMNPQSTWY), 595.5(ACDEFGHILMNQSTVW), N596(DEIPQT), 596.5(DENY), T597(DEHINQ), 597.5(F), Q598(ACEFHIMNVY), G599(CW), 600.5(F), L601(WY), P602(Q), 605.5(N), 606.5(P), 607.5(P), D608(W), R609(D), D610(Y), 610.5(F), 613.5(Y), Q614(T), I617(K), 617.5(1), P630(H), L633(FHY), 640.5(N), I646(M), L647(I), 657.5(C), S658(E), 658.5(C), T659(K), 659.5(CN), T660(H), F661(EQ), 661.5(C), S662(W), 662.5(FHLMW), 663.5(M), 664.5(C), 665.5(N), 667.5(Y), S674(G), 703.5(E), Y704(H), 704.5(DI), N705(EK), K706(DEGRST), S707(D), 707.5(MY), V708(CR), 708.5(1), V710(EN), 710.5(D), D711(N), 711.5(Y) 4.5(H), 10.5(1), E12(H), R20(Q), W23(M), 23.5(F), 24.5(N), P32(C), 0.8-fold A35(Y), 35.5(HY), 36.5(K), 38.5(ADGKV), K39(-AEV), 39.5(D), 40.5(DE), R43(GW), 45.5(M), G49(HM), Y50(H), 50.5(N), G58(C), 58.5(F), G62(Q), E63(CM), E74(I), A78(C), 78.5(Q), 83.5(DH), G86(D), D87(K), A98(N), E106(H), D107(Q), S109(F), 109.5(D), 117.5(K), F119(Q), 119.5(H), 122.5(E), 125.5(C), P128(F), 135.5(M), 143.5(K), R144(N), P150(M), 150.5(M), S156(T), 156.5(T), 157.5(P), T159(C), K161(DN), A162(—), G163(CN), Q164(M), 166.5(N), G174(C), G177(Q), 177.5(1), 179.5(CD), D180(CM), 180.5(CM), 181.5(NW), P183(C), 183.5(M), D184(Y), 184.5(MY), P185(EV), Q186(P), 186.5(C), P187(CK), 189.5(M), Q190(M), 191.5(W),P192(CMW), A193(C), 193.5(W), A194(C), P195(A), 195.5(Y), G197(A), L198(C), 199.5(CF), N201(D), M203(C), G206(F), P210(K), N215(DFM), G217(CEV), A218(Q), N223(CM), S225(C), H229(D), T233(D), 237.5(HQ), R238(H), 238.5(F), I240(CW), T244(C), P250(Q), N254(C), H255(N), L256(ACGHS), Q259(G), 260.5(ACDEMQST), S261(D), 261.5(S), S262(T), 262.5(EGINST), Q263(AGW), 263.5(DG), S264(-DEFLW), 264.5(D), 265.5(DE), 266.5(E), S267(ET), 267.5(E), N268(DEQ), 268.5(E), 269.5(D), N270(ACDGHSY), H271(EFI), F273(HMW), G280(A), F282(WY), H288(N), D295(E), R307(C), 307.5(K), P308(—), K309(CQ), L311(1), F313(C), K314(MV), T324(C), 324.5(CK), Q325(CHKR), N326(CLM), T330(KQ), 332.5(K), A333(C), V342(AI), T344(FI), S346(PW), Y348(DS), Q349(AGS), V353(T), G355(S), F365(W), A367(P), M371(F), T379(AG), 379.5(DE), L380(ENQTY), 380.5(E), N381(ACDGMQ), N382(DE), 382.5(D), 383.5(E), 384.5(D), Q385(PRT), 385.5(AP), A386(ELNRV), 386.5(CDEP), V387(CDE), 387.5(EGKP), R389(GIQY), S390(DGNT), P399(M), Q401(VW), L403(M), R404(K), 404.5(N), G406(DEN), N408(DGM), 408.5(CN), F409(CH), F411(Q), S412(EW), Y413(M), T414(G), 415.5(GMQ), E416(G), 416.5(EGNR), D417(I), 417.5(AKMQR), V418(GIM), P419(HILWY), Y424(C), 424.5(F), A425(-GQ), H426(N), S427(AC), Q428(EH), S429(CY), L430(M), D431(M), 431.5(Y), R432(-I), L433(DQ), 433.5(F), L437(AY), I438(GKT), D439(HKNPSTY), 441.5(M), L442(FI), 444.5(CLM), L445(CFMVY), 445.5(ACDEGHILMNQSTVY), S446(-CDEGHIKLMNQTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-ACDEFGHILMNPQSTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQSVWY), 448.5(ACDEFGHILMNPQSWY), N449(-ACDEFGILMPVW), 449.5(AEGHLMNPSTVY), T450(-CDEGPW), 450.5(ADEFGHLMPQSV), 451.5(GNP), S452(-DEFHNPVW), 452.5(AILPV), G453(-ADEFHIKLMNPQSTV), 453.5(FILMNTVY), T454(-DEFHILMNPV), 454.5(ADEFLMPSTY), T455(-DEILMQY), 455.5(AFHLPQTY), T456(-DEGHIKLMP), 456.5(ADEGHILMNPV), Q457(-DEFGLP), 457.5(ADEFGHILMNPVWY), S458(DEFHMNVWY), 458.5(DEFGHILMNPSTWY), R459(-ACDEFGHILMNPQSTVWY), 459.5(DEP), L460(AFHIMNPSTVWY), 460.5(W), Q461(DEIKV), F462(Y), 462.5(N), S463(ACDGKNQR), 463.5(F), Q464(ILV), 464.5(SY), A465(C), G466(CQ), 466.5(E), A467(HIMY), S468(CDFM), D469(V), 469.5(DE), I470(FL), R471(ACDEGKMQSTV), D472(MY), Q473(FHMSY), S474(I), R475(H), 475.5(SW), N476(TW), 476.5(L), W477(Y), 477.5(L), L478(FI), 478.5(L), P479(GIT), 479.5(D), G480(Y), P481(ACV), 481.5(GHY), C482(FGWY), 482.5(FM), Y483(ACFHIMTVW), 483.5(FQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACFGHILMNSTVY), 485.5(EIKMQY), Q486(ACDEFGILMNPSTVWY), 486.5(CFV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(AEFGHLMNQTWY), 488.5(ACDEFGILMPQSVWY), S489(-ACDEFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACDEFGHILMNPQRSTVWY), T491(-DEPY), 491.5(ACDEFGHIKLMNPQSTVWY), S492(-CDEFLM), 492.5(ADEGHILMPQSTVY), A493(-DEFHILNPQRVWY), 493.5(ADEFGHIKLMNPQSTVWY), D494(EFR), 494.5(ADEGHILMNPSTVWY), N495(-ADEFGHILMPQSTVWY), 495 5(ACDEFGILMPQSTVWY), N496(-ACDEFGHILMPQTVWY), 496.5(ADEFGHILMPQSTVWY), N497(-ACEFGHILMSVWY), 497.5(ADEFGILMPQVY), S498(-CDEFGILMNPQRTVWY), 498.5(ADEFGILMPQSVWY), E499(CDIPQS), 499.5(ACDEGHILMNPQRSTVWY), Y500(-ACDEGHIKLMNPQSTV), 500.5(ADEFGHILMNPQSTVWY), S501(-CDEFGILMNPTVWY), 501.5(ACDEFGHILMNQRSTVWY), W502(-ACDEFGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGILMNPQVWY), 503.5(ADEFGILMNPQSTVW), G504(-ACDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-AFGILMNPQV), 506.5(ACIPSTVW), K507(-ACEFHILMNQRSTVWY), Y508(-DS), H509(CEFNQ), 509.5(K), L510(I), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(CHIV), D514(ACEFGILMQSTV), S515(V), L516(ACIMSTV), V517(CIL), 517.5(C), N518(IQR), P521(FMNS), 521.5(MS), A522(CV), M523(CLV), 523.5(A), A524(GPS), 524.5(ACGHSTW), S525(ACGHMNP), 525.5(ACDEFGHILMNPQSTVWY), H526(-ACDEFGIKLMNPQSTVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILMPQRSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AEFMPQSVW), 528.5(DEPT), D529(S), 529.5(ADEGLMNPQTV), E530(NPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGHILMNPQSTVY), 531 5(ADEFGIKLMNPQRSTVY), K532(-ACDEFGHILMNPQRSTVWY), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHILMNQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-ACDEFGHNPTVWY), 536.5(ACILTV), S537(-ADFGNQY), 537.5(T), G538(—), V539(AHLMQST), 540.5(KMN), I541(-DGPT), 541.5(Q), F542(GP), 542.5(QY), G543(P), 543.5(ACDIKPQRTVY), K544(-FGMNPQ), 544.5(ADEFGHIKLMRVWY), Q545(-FK), 545.5(ACFILMNPQSVWY), G546(DEHLPTVWY), 546.5(ACEGHILMNPQSTVWY), S547(-DHIMQY), 547.5(ADGHNTVWY), E548(FNY), 548.5(ACDEFGHLMNPTWY), K549(-DFHILTWY), 549.5(ADFHLMQSTV), T550(CW), 550.5(AIKMNPRSTV), N551(ACDEHIKLPQRTV), 551.5(ACDEHILMNS), V552(N), D553(AGST), I554(EHMPQS), E555(AGHNSTVY), 555.5(N), K556(ACDEFGHMQTW), V557(L), M558(ADNSTV), 558.5(E), 1559(C), 559.5(A), T560(AV), 560.5(A), D561(EFLY), E562(AD), E563(FITVY), I565(T), R566(ACDEFGHILMNQSTVWY), T567(Q), 567.5(E), T568(PV), 569.5(M), P570(CQS), 570.5(M), V571(ACDEFHLMQSTY), 571.5(G), A572(CGSY), 572.5(VWY), T573(ACDGINSVW), 573.5(MT), E574(ADLMQS), 574.5(DV), Q575(-CDGIKLMNSTVWY), 575.5(AFWY), Y576(ACFGHIMSV), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(DEFGHILNVWY), 578.5(ACDEFGHILNSTVY), V579(ACDEFGHLMNQSTWY), 579.5(ACDEFGILMNPQSTVWY), S580(-DEFHILMNPQTVWY), 580.5(ACDEFGKLMNPQRSTVWY), T581(-CDEFGHKLNPWY), 581 5(ACDEFGILMNPQTVWY), N582(-ACDEFGHIKLMPQRSTVWY), 582.5(DFGHNPSTVW), L583(-ADEGIPQST), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMNPQSTVWY), R585(-ACDEFGHIKLMNPQSTVWY), 585.5(ADEFGHILMNPQSTVWY), G586(-DEFHLMNSVWY), 586.5(ACDEFGHILMNPQSTVY), N587(-DEFGHILMPQSTVY), 587.5(ADEFGILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(ADEFGHILMNPQSTVY), Q589(-DEIPRW), 589.5(ACDEFGHIKLMNPQSTVWY), A590(DEFHIKLMNQTWY), 590.5(ACDEFGIKLMNPQSTVWY), A591(-CDEFGILMNPTVWY), 591.5(ACDEFGHILMNPQSTVWY), T592(-ACDEFGHLMNPQSVY), 592.5(ACDEFGILMNPQSTVWY), A593(-DEP), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(ACDEFGHLMNPQSTWY), 595.5(ACDEFGHILMNQSTVW), N596(ACDEGIMPQTW), 596.5(DENY), T597(DEHINPQV), 597.5(F), Q598(ACEFGHILMNSTVY), G599(CSW), 600.5(F), L601(WY), P602(MQ), M604(AS), 605.5(N), 606.5(P), 607.5(P), D608(W), R609(D), D610(Y), 610.5(F), Y612(R), 613.5(Y), Q614(T), I617(K), 617.5(I), K620(HMN), H623(L), F628(HIM), P630(GHI), L633(FHY), F637(Y), K640(W), 640.5(N), P644(W), I646(M), L647(I), N650(L), P652(S), 657.5(C), S658(CE), 658.5(CKR), T659(K), 659.5(CNQY), T660(CHP), 660.5(CFIW), F661(EQT), 661.5(CP), S662(W), 662.5(FHLMPQW), 663.5(CIMW), 664.5(C), K665(I), 665.5(NW), 666.5(V), A667(C), 667.5(HY), S668(N), T671(R), Q672(E), S674(G), V678(T), S679(EGIKY), E681(M), E683(H), E685(ALS), L686(T), Q687(I), 697.5(N), 701.5(A), N703(D), 703.5(EHLTY), Y704(H), 704.5(ADILNS), N705(DEK), 705.5(EQY), K706(DEGRST), S707(DEH), 707.5(MY), V708(CER), 708.5(I), N709(DER), 709.5(CGP), V710(EMNP), 710.5(D), D711(GN), 711.5(Y), T716(C), 720.5(N), E722(D), P723(CEQ), R724(W), P725(M) A2(C), 3.5(DNTV), 4.5(DGHY), G5(Y), D9(G), W10(Y), 10.5(HI), 0.9-fold E12(HIK), L15(H), S16(Q), I19(T), R20(GINQ), Q21(GK), 21.5(F), 22.5(Y), W23(DFKM), 23.5(F), K24(C), 24.5(DIN), L25(GR), 25.5(CVW), P27(T), 28.5(AEGINV), P29(ACEF), P30(Y), 30.5(GIKN), P31(H), 31.5(AY), P32(CIKS), P34(Q), 34.5(CV), A35(PY), 35.5(CGHNY), E36(CQ), 36.5(K), R37(F), 37.5(EMP), H38(ADE), 38.5(ADEFGKVY), K39(-AEPV), 39.5(DEHN), D40(-PY), 40.5(DEST), D41(A), 41.5(NQ), S42(-EHLQV), 42.5(KY), R43(GW), 43.5(DVY), 45.5(M), G49(HMQY), Y50(H), 50.5(N), Y52(D), 53.5(R), P55(Y), 55.5(E), F56(AV), G58(CT), 58.5(F), L59(CIR), 59.5(H), D60(NY), K61(C), 61.5(K), G62(Q), E63(CMV), E67(PV), A70(I), A71(NTY), L73(KNVY), E74(DFI), A78(CKQ), 78.5(AQ), Y79(—), R81(K), Q82(-HKM), 82.5(DK), L83(N), 83.5(DHNY), D84(IT), 84.5(N), S85(D), 85.5(HK), G86(-DH), 86.5(Q), D87(EKQ), 87.5(AHQ), N88(AI), 90.5(Y), L91(QY), K92(N), 92.5(D), Y93(G), N94(Q), D97(—), A98(N), 98.5(E), E99(DK), 99.5(K), Q101(DY), L104(C), 104.5(E), K105(MV), 105.5(HRW), E106(HN), 106.5(WY), D107(-QW), 107.5(EP), 108.5(W), S109(FKY), 109.5(D), F110(VY), 112.5(C), N113(CDW), 113.5(FN), LI 14(HNQ), G115(M), 115.5(A), 116.5(DEP), A117(NQY), 117.5(CKW), VI 18(K), F119(QY), 119.5(H), Q120(N), 120.5(K), 121.5(K), K122(-DFR), 122.5(EIKP), K123(CENW), 123.5(N), R124(—), 124.5(EK), 125.5(C), 126.5(A), E127(-F), 127.5(DIY), P128(F), 128.5(T), L129(CKW), G130(C), 130.5(D), L131(NV), 131.5(EHR), E133(FW), E134(K), 134.5(A), P135(Q), 135.5(DM), V136(Y), 136.5(CM), 137.5(Q), 139.5(R), P140(H), 140.5(N), G141(CK), 141.5(Q), K142(CH), 142.5(CEGW), 143.5(K), R144(NW), 144.5(RY), P145(AR), 145.5(KMY), 146.5(N), 147.5(EH), H148(CNW), 148.5(EMN), S149(W), 149.5(HM), P150(-M), 150.5(CDM), V151(D), E152(-DH), 152.5(CHK), P153(W), S156(T), 156.5(T), S157(EKNY), 157.5(CNPQ), G158(D), T159(C), 159.5(V), G160(C), 160.5(CDN), K161(CDHNTWY), 161.5(EHN), A162(-H), 162.5(ACSVY), G163(CFNPT), 163.5(CN), Q164(DM), 164.5(CEY), Q165(HW), 165.5(ITV), P166(AILS), 166.5(GN), N172(Y), 173.5(FI), G174(CFHS), Q175(DKT), 175.5(CILPRTW), T176(CGNRS), 176.5(GN), G177(-EQ), 177.5(IP), D178(NQW), 178.5(DGINW), 179.5(CDN), D180(CM), 180.5(ACM), S181(QRWY), 181.5(EIKLNPWY), V182(CGNW), 182.5(CFLPRW), P183(CS), 183.5(EIMY), D184(HNVY), 184.5(MY), P185(-DEIV), 185.5(D), Q186(NP), 186.5(CIK), P187(-CHK), 187.5(V), 188.5(Y), G189(—), 189.5(MQ), Q190(MTW), 190.5(DFMN), P191(CQ), 191.5(W), P192(CKMQW), 192.5(CMN), A193(CDFLM), 193.5(DGHLMNPW), A194(CKMRW), 194.5(CDM), P195(AKMNQR), 195.5(EMQY), S196(-HW), G197(AT), L198(-CDGINRV), G199(V), 199.5(CFHW), T200(IQV), N201(DHV), T202(NS), M203(ACDPR), A204(D), T205(IKR), G206(DEF), S207(D), G208(E), A209(ENP), P210(KQS), D213(LTW), N214(CEILY), N215(DFIM), E216(A), G217(CEFHLMVY), A218(PQ), G220(CNT), V221(I), N223(CHMY), S225(CQR), N227(D), H229(DSTV), D231(C), T233(DHL), M235(V), G236(V), 236.5(D), D237(C), 237.5(DGHKNQS), R238(CH), 238.5(FT), I240(CRW), T242(R), T244(C), A248(V), L249(V), P250(Q), T251(LV), N254(CGQ), H255(ANT), L256(ACGHMNQRSV), K258(LQ), Q259(CGHKPRS), I260(M), 260.5(ACDEMQST), S261(CDG), 261.5(S), S262(T), 262.5(DEGINST), Q263(-ADFGHWY), 263.5(DG), S264(-DEFGHLWY), 264.5(D), G265(—), 265.5(ADET), 266.5(AE), S267(ENT), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(EFIN), F273(HMWY), S276(ACT), G280(A), Y281(F), F282(WY), F287(Y), H288(N), C289(A), D295(AE), R307(CNST), 307.5(K), P308(-W), K309(ACQS), R310(FILW), L311(AFIY), N312(LM), F313(CI), K314(CGHLMQSVWY), 1318(F), T324(CL), 324.5(CKR), Q325(CHIKRV), 325.5(1), N326(CDLM), 326.5(M), D327(T), G328(AHNS), T329(ACI), T330(KQ), T331(ALY), I332(ACS), 332.5(K), A333(CD), N334(Q), T339(C), V342(AIL), T344(DFILM), S346(FIPW), Y348(ADMNRS), Q349(AGS), V353(CT), L354(AHT), G355(AS), S356(A), Q359(EGHM), F365(W), A367(P), V369(P), F370(Y), M371(AFS), V372(C), Q374(N), Y375(W), Y377(F), L378(A), T379(ACGV), 379.5(DE), L380(ENQTY), 380.5(E), N381(ACDGMQS), N382(CDE), 382.5(D), 383.5(E), 384.5(D), Q385(ENPRSTW), 385.5(AEHPT), A386(EHLNPRTVW), 386.5(CDEP), V387(CDELM), 387.5(EGKP), G388(IM), R389(AGILMPQSTVY), 389.5(N), S390(DGNT), S391(A), 394.5(N), Y397(AN), P399(EIMQ), Q401(CDIMRVW), L403(M), R404(K), 404.5(N), T405(S), G406(DEN), N408(DGHLM), 408.5(CN), F409(CHY), T410(H), F411(LQSTV), S412(CEFGILW), Y413(MW), T414(FGQ), 414.5(D), 415.5(ADGHMNQS), E416(AGP), 416.5(CEGMNR), D417(EFHILMNS), 417.5(ACEHKLMQRT), V418(GIMQST), P419(DEGHIKLNQVWY), F420(L), Y424(CT), 424.5(F), A425(-GQV), H426(N), S427(ACD), Q428(EHNS), S429(CPY), L430(IM), D431(FM), 431.5(Y), R432(-I), L433(DIQV), 433.5(F), M434(A), L437(ACHMNSTY), I438(GKNT), D439(HKNPSTY), 441.5(M), L442(CFIT), Y444(FH), 444.5(CLM), L445(CFMVY), 445.5(ACDEGHILMNQSTVY), S446(-CDEGHIKLMNQTV), 446.5(ACDEFGHILMNPQSTVWY), R447(-ACDEFGHILMNPQSTVWY), 447.5(ACDEFGHILMNPQSVWY), T448(-ACDEFGHILMNPQSVWY), 448.5(ACDEFGHILMNPQSWY), N449(-ACDEFGILMPQSTVWY), 449.5(AEGHLMNPSTVY), T450(-CDEGHPQW), 450.5(ADEFGHLMPQSTV), P451(-I), 451.5(GNP), S452(-DEFHILNPQVW), 452.5(AILPV), G453(-ADEFHIKLMNPQSTV), 453.5(FILMNTVY), T454(-DEFHILMNPV), 454.5(ADEFGLMPSTY), T455(-ADEILMNPQVY), 455.5(AFHLNPQSTY), T456(-DEFGHIKLMNPQ), 456.5(ADEGHILMNPQSV), Q457(-ADEFGHILP), 457.5(ADEFGHILMNPSVWY), S458(-CDEFHMNTVWY), 458.5(CDEFGHILMNPSTVWY), R459(-ACDEFGHILMNPQSTVWY), 459.5(DEP), L460(ACFHIMNPSTVWY), 460.5(W), Q461(CDEIKTVW), F462(Y), 462.5(N), S463(ACDGKNQRTV), 463.5(F), Q464(AFILRSTV), 464.5(SY), A465(CGP), G466(CQ), 466.5(E), A467(DFGHIKLMRVY), S468(CDEFLMQRV), D469(V), 469.5(DE), I470(FLM), R471(ACDEGKMQSTV), D472(LMY), Q473(FHMSY), S474(IY), R475(H), 475.5(SW), N476(CSTW), 476.5(L), W477(Y), 477.5(L), L478(FI), 478.5(L), P479(GIT), 479.5(D), G480(Y), P481(ACV), 481.5(GHY), C482(FGIMNQTWY), 482.5(FM), Y483(ACFHIMTVW), 483.5(FQV), R484(EFGHIKLMNQSTVY), 484.5(N), Q485(ACFGHILMNSTVY), 485.5(EIKMQY), Q486(ACDEFGILMNPSTVWY), 486.5(CFV), R487(-ACDEFGHIKLMNPQSTVWY), 487.5(FW), V488(ACEFGHLMNQTWY), 488.5(ACDEFGILMPQSVWY), S489(-ACDEFGHIKLMNPQTVWY), 489.5(ACDEGLMPQRSTVWY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACDEFGHILMNPQRSTVWY), T491(-ADEGPSWY), 491.5(ACDEFGHIKLMNPQSTVWY), S492(-CDEFGILMNQ), 492.5(ADEGHILMPQSTVY), A493(-DEFHILNPQRVWY), 493 5(ADEFGHIKLMNPQSTVWY), D494(EFR), 494.5(ADEGHILMNPSTVWY), N495(-ADEFGHILMPQSTVWY), 495.5(ACDEFGILMPQSTVWY), N496(-ACDEFGHILMPQSTVWY), 496.5(ADEFGHILMPQSTVWY), N497(-ACEFGHILMPSTVWY), 497.5(ADEFGILMPQTVY), S498(-CDEFGILMNPQRTVWY), 498.5(ADEFGILMNPQSTVWY), E499(CDIPQS), 499.5(ACDEGHILMNPQRSTVWY), Y500(-ACDEGHIKLMNPQSTV), 500.5(ADEFGHILMNPQSTVWY), S501(-ACDEFGILMNPQRTVWY), 501.5(ACDEFGHILMNQRSTVWY), W502(-ACDEFGILMPQSTVY), 502.5(ADEFGHILMNPQSTVWY), T503(-ADEFGHILMNPQVWY), 503.5(ADEFGILMNPQSTVW), G504(-ACDEFHILMNPQSTVWY), 504.5(ACDEFGHILMNPQSTVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACDEFGILMPQSTVWY), T506(-ACFGILMNPQV), 506.5(ACIPSTVW), K507(-ACEFHILMNQRSTVWY), Y508(-DMST), H509(CEFNQ), 509.5(K), L510(I), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(CHIV), D514(ACEFGILMQSTV), S515(CV), L516(ACIMSTV), V517(CIL), 517.5(C), N518(CILQRTV), P521(CFLMNST), 521.5(MS), A522(CV), M523(CLQV), 523.5(A), A524(CGPST), 524.5(ACGHSTW), S525(ACGHMNPT), 525.5(ACDEFGHILMNPQSTVWY), H526(-ACDEFGIKLMNPQSTVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-DEFGHILMPQRSTVWY), 527.5(ACDEFGHILMPQSTVWY), D528(AEFMPQSVW), 528.5(DEPT), D529(ES), 529.5(ADEGLMNPQTV), E530(DNPVW), 530.5(ACDEFGHIKLMNQRSTVWY), E531(ACFGHILMNPQSTVY), 531.5(ADEFGIKLMNPQRSTVY), K532(-ACDEFGHILMNPQRSTVWY), 532.5(ACDEFGHIKLMNPQSTVWY), F533(ACDEGHILMNQSTVW), 533.5(ACDEFGHILNPQSTVWY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-ACDEFGHIMNPSTVWY), 536.5(ACILTV), S537(-ACDFGNQTY), 537.5(T), G538(—), V539(AHILMNQST), L540(IM), 540.5(EKMN), I541(-ADEFGPQRTVY), 541.5(DEGPQST), F542(GKP), 542.5(QY), G543(FPS), 543.5(ACDHIKLMPQRSTVY), K544(-ACFGHILMNPQSTVY), 544.5(ACDEFGHIKLMRSTVWY), Q545(-ADFKLPVW), 545.5(ACDFGHILMNPQSTVWY), G546(ACDEFHILMNPQSTVWY), 546.5(ACDEFGHILMNPQSTVWY), S547(-DEFGHILMPQWY), 547.5(ACDEFGHILMNPQSTVWY), E548(FILNPQY), 548.5(ACDEFGHILMNPSTWY), K549(-DFGHILPQSTVWY), 549.5(ADEFGHLMNQSTV), T550(-ACDRW), 550.5(ADIKMNPRSTV), N551(ACDEGHIKLMPQRSTVY), 551 5(ACDEHILMNPSTV), V552(ACELNST), 552.5(VW), D553(AGST), I554(ACEFHLMPQSTW), E555(AGHNSTVY), 555.5(N), K556(ACDEFGHLMQSTVW), 556.5(Q), V557(L), M558(ACDINQSTVWY), 558.5(E), I559(CQV), 559.5(A), T560(ACSV), 560.5(A), D561(AEFLQRY), E562(AD), E563(AFHIMRTVY), I565(T), R566(ACDEFGHILMNQSTVWY), T567(PQ), 567.5(E), T568(PV), 569.5(M), P570(CQS), 570.5(M), V571(ACDEFHILMQSTY), 571.5(G), A572(CGSY), 572.5(VWY), T573(ACDGHINSVW), 573.5(MT), E574(ACDFLMQSW), 574.5(DV), Q575(-ACDFGHIKLMNSTVWY), 575.5(AFWY), Y576(ACFGHIMSVW), 576.5(EFWY), G577(DW), 577.5(ACDEHIMNQSTV), S578(DEFGHILNQVWY), 578.5(ACDEFGHILNSTVY), V579(ACDEFGHLMNQSTWY), 579.5(ACDEFGILMNPQSTVWY), S580(-DEFHILMNPQTVWY), 580.5(ACDEFGHIKLMNPQRSTVWY), T581(-CDEFGHKLNPWY), 581.5(ACDEFGILMNPQTVWY), N582(-ACDEFGHIKLMPQRSTVWY), 582.5(DFGHNPSTVW), L583(-ADEGIPQSTVW), 583.5(DEGIMNPQSTVY), Q584(-ADEFGHILMNPSTVWY), 584.5(ACDEFGHILMNPQSTVWY), R585(-ACDEFGHIKLMNPQSTVWY), 585.5(ADEFGHILMNPQSTVWY), G586(-DEFHLMNQSTVWY), 586.5(ACDEFGHILMNPQSTVY), N587(-DEFGHILMPQSTVY), 587.5(ADEFGILMNPQSTVWY), R588(-ACDEFGHIKLMNPQSTVWY), 588.5(ADEFGHILMNPQSTVY), Q589(-ADEFIPRSTVW), 589.5(ACDEFGHIKLMNPQSTVWY), A590(DEFGHIKLMNQTWY), 590.5(ACDEFGIKLMNPQSTVWY), A591(-CDEFGILMNPQTVWY), 591.5(ACDEFGHILMNPQSTVWY), T592(-ACDEFGHLMNPQSVY), 592.5(ACDEFGILMNPQSTVWY), A593(-DELPY), 593.5(ACDEFGHILMNPQSTVWY), D594(ELT), 594.5(ACDEFGHILMNPQSTVWY), V595(ACDEFGHLMNPQSTWY), 595.5(ACDEFGHILMNQSTVW),N596(ACDEFGIMPQSTWY), 596.5(DENY), T597(DEGHINPQRVW), 597.5(F), Q598(ACEFGHILMNSTVY), G599(CSW), 600.5(F), L601(VWY), P602(CMQ), M604(AQS), V605(N), 605.5(N), 606.5(P), 607.5(P), D608(W), R609(D), D610(Y), 610.5(F), Y612(R), 613.5(Y), Q614(CT), I617(K), 617.5(1), A619(CG), K620(ACHMNQS), I621(AHLR), H623(EKL), D625(S), G626(A), H627(VY), F628(HILM), P630(AGHIQSTV), 630.5(C), S631(N), L633(FHMY), M634(C), F637(IWY), L639(V), K640(FPVW), 640.5(N), H641(C), P644(WY), Q645(ILMTV), I646(MT), L647(I), I648(AC), N650(LTV), P652(AS), V653(T), 655.5(IW), N656(FT), 657.5(CDHIS), S658(CEFKMQ), 658.5(CFGHKLMQRW), T659(DKRS), 659.5(CELMNQRY), T660(-CFHKLP), 660.5(CEFHIPRW), F661(AEILQT), 66E5(CDELPQRSW), S662(NPW), 662.5(ACDFHKLMPQTVW), A663(HY), 663.5(CDEGIMNW), A664(CEHK), 664.5(CDGPRTV), K665(CGILSVWY), 665.5(HIMNPRVW), F666(C), 666.5(IVY), A667(CP), 667.5(HQY), S668(N), F669(Y), I670(V), T671(R), Q672(EL), S674(G), T675(AL), Q677(C), V678(AIT), S679(EFGHIKMRY), V680(L), E681(LMNT), I682(MV), E683(HILM), E685(ADLST), L686(CT), Q687(FIKMW), E697(-DLPR), 697.5(CGHNPSW), I698(V), 699.5(E), Y700(F), T701(S), 701.5(A), S702(CNV), 702.5(Q), N703(D), 703.5(AEHLTY), Y704(H), 704.5(ADEILNST), N705(CDEKMPW), 705.5(ENQY), K706(DEGRST), S707(CDEFH), 707.5(MY), V708(CEFGHMNRW), 708.5(1), N709(CDEGPR), 709.5(CGPQ), V710(EMNPQ), 710.5(D), D711(AGNP), 711.5(QY), T713(C), T716(CEFMPRVWY), N717(ER), V719(CFIKNR), 720.5(N), S721(FKLMQRTVW), E722(DQ), P723(CDEQSV), R724(FHSW), P725(ALMQ), I726(V), L735(CI), 735.5(LMV)

TABLE 9 Amino acid alterations that reduce delivery to blood. Fold increase of Amino acid alteration efficiency S264(W), 444.5(M), S446(D), R471(G), P479(G), 479.5(D), C482(Y), 0.1-fold Y483(ACHV), Q485(AGS), 485.5(K), 487.5(F), 488.5(W), 502.5(Y), 504.5(NW), A505(Y), 505.5(AQTVW), T506(FL), 506.5(AV), N518(R), 524.5(S), 525.5(ANQTY), 526.5(FHMY), 527.5(HW), Q536(—), S537(—), G538(—), V539(H), A572(Y), T573(G), Y576(ACGH), V579(G), V595(DGHNQY), 595.5(H), Q598(GNY), L601(W) S264(W), 266.5(E), 268.5(E), 379.5(E), 444.5(M), S446(D), N449(C), 0.2-fold 454.5(E), R459(C), F462(Y), 469.5(E), R471(AEGQSTV), 475.5(W), P479(GT), 479.5(D), G480(Y), C482(GY), Y483(ACHIQTV), R484(K), 484.5(N), Q485(AGNST), 485.5(K), 487.5(F), V488(G), 488.5(PW), 491.5(W), S492(C), 498.5(DE), 499.5(ADLMNPQ), 500.5(LNQST), 501.5(ACHNS), W502(-AE), 502.5(AGMNPTY), 503.5(AGILMNQT), G504(W), 504.5(AGHMNQVWY), A505(HLY), 505.5(AGLMQTVW), T506(FGILMP), 506.5(ACISV), K507(HIW), H509(E), D514(GIV), N518(R), P521(M), 523.5(A), 524.5(AS), S525(H), 525.5(AFHILNQSTY), H526(IPV), 526.5(AFHMNQSTY), 527.5(CFHIMQVWY), 530.5(C), 531.5(I), 532.5(P), F534(VY), P535(-F), Q536(-P), S537(-DFQ), G538(—), V539(H), P570(Q), A572(Y), T573(AG), E574(F), Q575(L), Y576(ACGHS), S578(D), V579(EGMNQ), S580(Q), N582(F), Q589(P), V595(DEGHLMNPQTWY), 595.5(ADHIQSTW), 596.5(N), T597(GLP), Q598(CEFGNSY), G599(C), L601(W), 640.5(N) S264(W), 266.5(E), 267.5(E), 268.5(E), N270(AGHSY), 379.5(E), 380.5(E), 0.3-fold 431.5(Y), 433.5(F), 444.5(CLM), L445(Y), 445.5(ACLMSTY), S446(DEIL), 446.5(ALPQ), R447(I), 447.5(PW), T448(ILPQW), 448.5(CP), N449(CP), S452(D), 454.5(DE), T455(E), Q457(E), S458(D), 458.5(CW), R459(CDV), 460.5(W), Q461(D), F462(Y), D469(M), 469.5(E), R471(ACEGMQSTV), Q473(Y), S474(I), 475.5(SW), 477.5(L), P479(GT), 479.5(D), G480(Y), C482(FGY), 482.5(M), Y483(ACHIQTV), 483.5(Q), R484(K), 484.5(N), Q485(ACGNSTV), 485.5(KQY), 487.5(F), V488(FGY), 488.5(MPQW), S489(FQY), K490(DEFGHNQWY), 490.5(CPQ), 491.5(W), S492(C), 495.5(W), N496(M), S498(DE), 498.5(DE), 499.5(ADEGLMNPQST), Y500(-NP), 500.5(AGLMNPQST), S501(-CIL), 501.5(ACGHILNQSTVY), W502(-ACEV), 502.5(AFGILMNPSTVY), T503(EP), 503.5(AGILMNPQSTVW), G504(-IPW), 504.5(AFGHILMNPQSVWY), A505(FHILMPWY), 505.5(AGILMPQSTVWY), T506(FGILMNP), 506.5(ACISTV), K507(ACHINQSW), H509(EFN), N511(E), R513(C), D514(AGIV), L516(AST), 517.5(C), N518(R), P521(M), 523.5(A), 524.5(AS), S525(HM), 525.5(ACFHILMNQSTVWY), H526(-EGIKMPQV), 526.5(ACFGHLMNQSTVY), 527.5(ACDEFGHILMPQSTVWY), 528.5(DE), 529.5(DP), E530(P), 530.5(CILM), 531.5(IV), 532.5(P), F533(T), F534(VY), P535(-F), Q536(-DPW), S537(-DFGNQY), G538(—), V539(H), 559.5(A), T568(P), P570(Q), 570.5(M), V571(AHQS), A572(Y), T573(ACDG), E574(FLW), Q575(DGL), 575.5(W), Y576(ACGHS), 576.5(W), 577.5(HS), S578(DEFGL), 578.5(L), V579(EGHMNQ), S580(Q), N582(FWY), 585.5(A), 586.5(P), Q589(P), 593.5(P), V595(ACDEGHLMNPQTWY), 595.5(ADFHILMQSTW), 596.5(DN), T597(EGILMPV), Q598(CEFGNSTY), G599(C), L601(WY), L633(FY), 640.5(N) D180(M), 183.5(M), L256(S), S261(D), Q263(V), S264(W), 265.5(DE), 0.5-fold 266.5(E), S267(E), 267.5(E), 268.5(E), 269.5(D), N270(ACDGHSY), H271(E), 379.5(DE), L380(E), 380.5(E), N382(D), 382.5(D), 383.5(E), 386.5(D), Q428(E), D431(M), 431.5(Y), R432(I), L433(D), 433.5(F), L437(Y), Y444(FH), 444.5(CLM), L445(FY), 445.5(ACGHLMQSTVY), S446(DEILV), 446.5(AFILPQSTVWY), R447(CEGILMQTV), 447.5(CFGIMPWY), T448(FGILNPQVWY), 448.5(CFLPW), N449(CDEFP), T450(CE), S452(D), 454.5(DE), T455(E), 456.5(D), Q457(DE), 457.5(D), S458(CDEW), 458.5(CDEMWY), R459(ACDEILMPQSTV), L460(NSTWY), 460.5(W), Q461(DE), F462(Y), S463(G), 463.5(F), Q464(KSV), 464.5(S), A465(CGP), A467(GY), S468(C), D469(CEMQRSV), 469.5(E), I470(FL), R471(ACDEGMQSTV), Q473(FMY), S474(I), 475.5(SW), N476(TW), 477.5(L), L478(F), P479(GT), 479.5(D), G480(Y), 481.5(Y), C482(FGY), 482.5(FM), Y483(ACHILMQTV), 483.5(FQ), R484(K), 484.5(N), Q485(ACGHILNSTVY), 485.5(EKQY), Q486(FIV), 487.5(FW), V488(EFGHLNWY), 488.5(FILMPQW), S489(FGILQVY), 489.5(DP), K490(ACDEFGHILMNQSTVWY), 490.5(CIPQV), T491(E), 491.5(FIMW), S492(CE), 492.5(DL), A493(DE), 493.5(KP), N495(AFLQTVY), 495.5(FILPVW), N496(AFGHILMW), N497(-I), 497.5(DE), S498(-DELMR), 498.5(DEGLQ), E499(I), 499.5(ADEGHILMNPQSTV), Y500(-ACEGKLNPQS), 500.5(AFGHILMNPQST), S501(-CILNV), 501.5(ACFGHILMNQRSTVWY), W502(-ACEGILMQSVY), 502.5(AFGHILMNPQSTVWY), T503(-ADEP), 503.5(AFGILMNPQSTVW), G504(-CILNPSVWY), 504.5(AFGHILMNPQSVWY), A505(EFHILMNPQVWY), 505.5(ACFGILMPQSTVWY), T506(FGILMNPV), 506.5(ACISTV), K507(ACHILMNQSVW), Y508(—), H509(EFN), L510(I), N511(DELMW), 512.5(S), R513(CIV), D514(ACGILMQTV), L516(ACSTV), V517(L), 517.5(C), N518(LR), P521(CFLMNS), 521.5(S), 523.5(A), 524.5(ACST), S525(HMP), 525.5(ACFGHILMNQSTVWY), H526(-ACDEFGIKMNPQSTVWY), 526.5(ACDEFGHLMNPQSTVY), K527(—), 527.5(ACDEFGHILMPQSTVWY), 528.5(DEPT), 529.5(ADEMNPTV), E530(P), 530.5(CDEFGHILMNQSTVWY), 531.5(ADFGINPQTVY), 532.5(HPSTW), F533(ACILQSTV), 533.5(Y), F534(ILVY), 534.5(F), P535(-AF), Q536(-DPWY), S537(-CDFGNQTY), G538(—), V539(H), 542.5(Y), G543(S), 543.5(Y), K544(FNQY), S547(—), K549(IW), 549.5(E), T550(CD), N551(DE), 55E5(DE), V552(NS), I554(E), V557(L), 558.5(E), 559.5(A), R566(ADEFGILMNTVWY), 567.5(E), T568(P), 569.5(M), P570(Q), 570.5(M), V571(ACHMQS), 571.5(G), A572(CY), T573(ACDGNSV), 573.5(M), E574(DFLMW), 574.5(V), Q575(DGKLMNV), 575.5(AWY), Y576(ACGHIS), 576.5(FWY), 577.5(AHMNS), S578(DEFGILWY), 578.5(AGHLNST), V579(ACEFGHMNQST), 579.5(L), S580(-MNQ), T581(ENP), N582(FILWY), 585.5(AGIMPQSTVY), 586.5(AGPQ), 587.5(MNQV), 588.5(LMV), Q589(APV), 589.5(PV), 590.5(CP), A591(DT), 591.5(FILP), T592(-DGN), 592.5(CGMS), A593(-D), 593.5(APSW), D594(E), 594.5(AGQST), V595(ACDEFGHLMNPQSTWY), 595.5(ADEFHILMQSTW), N596(CETW), 596.5(DEN), T597(DEGIKLMPQRV), Q598(ACEFGHNSTY), G599(C), L601(WY), P602(M), M604(A), 605.5(N), 607.5(P), 610.5(F), L633(FHY), 640.5(N), 662.5(W) D41(H), 41.5(D), S42(E), R43(T), 43.5(DT), 45.5(M), K51(Y), G58(ET), 0.7-fold K61(—), E63(M), 107.5(E), D180(M), 180.5(M), 183.5(M), L256(ACS), 260.5(AEM), S261(D), S262(T), 262.5(DE), Q263(VW), S264(DEW), 264.5(D), 265.5(DE), 266.5(E), S267(E), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(E), R307(MN), P308(M), 324.5(C), 326.5(M), Q359(DET), 379.5(DE), L380(E), 380.5(E), N381(D), N382(DE), 382.5(D), 383.5(E), 384.5(D), A386(E), 386.5(CDE), V387(E), 387.5(P), G388(E), 394.5(N), 408.5(C), 424.5(F), Q428(EH), S429(Y), D431(M), 431.5(Y), R432(I), L433(D), 433.5(F), L437(HY), L442(FI), Y444(FH), 444.5(CLM), L445(CFMY), 445.5(ACGHLMNQSTVY), S446(CDEGHILMTV), 446.5(AFHILNPQSTVWY), R447(ACEFGILMNQSTV), 447.5(CFGIMPVWY), T448(CFGILMNPQVWY), 448.5(CEFILPWY), N449(ACDEFGIMPSVW), T450(CE), S452(DE), T454(DEFI), 454.5(DE), T455(DE), T456(D), 456.5(D), Q457(DEIL), 457.5(DE), S458(CDEFMPVW), 458.5(CDEFIMWY), R459(-ACDEFGHILMNPQSTVWY), L460(ANSTVWY), 460.5(W), Q461(DEGK), F462(Y), S463(CDGNQ), 463.5(F), Q464(ACIKLSTV), 464.5(S), A465(CGP), G466(AQ), A467(DFGPQY), S468(CP), D469(ACEMQRSTV), 469.5(DE), I470(FLM), R471(ACDEGKMQSTV), D472(Y), Q473(FHMY), S474(I), R475(K), 475.5(SW), N476(TW), W477(Y), 477.5(L), L478(FI), P479(GIT), 479.5(D), G480(Y), P481(A), 481.5(HY), C482(FGY), 482.5(FM), Y483(ACHILMQTV), 483.5(FQ), R484(K), 484.5(N), Q485(ACFGHILNSTVY), 485.5(EKQY), Q486(FGINSVWY), 486.5(F), R487(H), 487.5(FW), V488(AEFGHLMNQTWY), 488.5(CFGILMPQVWY), S489(-ACFGHIKLPQTVY), 489.5(DEP), K490(-ACDEFGHILMNQRSTVWY), 490.5(ACFHINPQTV), T491(DE), 491.5(ACFILMPQTVWY), S492(CDELM), 492.5(ADEGILV), A493(DELV), 493.5(IKLMPV), D494(R), 494.5(GIPV), N495(-AFLMPQSTVY), 495.5(AFGILPSVWY), N496(-ACFGHILMQSVW), N497(-EITV), 497.5(DEGLM), S498(-ADEFGILMPQRVWY), 498.5(DEFGLMQY), E499(I), 499.5(ADEGHILMNPQRSTVWY), Y500(-ACDEGIKLNPQST), 500.5(AEFGHILMNPQSTW), S501(-ACILMNPTVW), 501.5(ACFGHILMNQRSTVWY), W502(-ACEGILMPQSVY), 502.5(AFGHILMNPQSTVWY), T503(-ADEFGIPV), 503.5(AFGILMNPQSTVW), G504(-ACFILMNPSTVWY), 504.5(ACFGHILMNPQSVWY), A505(-EFHILMNPQTVWY), 505.5(ACFGILMPQSTVWY), T506(ACFGILMNPQV), 506.5(ACIPSTVW), K507(ACHILMNQSTVW), Y508(-D), H509(CEFN), 509.5(K), L510(I), N511(DELMQWY), 512.5(S), R513(CIV), D514(ACEGILMQSTV), L516(ACIMSTV), V517(L), 517.5(C), N518(ILQRTV), P521(CFLMNS), 521.5(S), A522(CV), 523.5(A), A524(CGP), 524.5(ACST), S525(ACHMP), 525 5(ACFGHILMNQSTVWY), H526(-ACDEFGIKLMNPQRSTVWY), 526.5(ACDEFGHILMNPQSTVY), K527(-HR), 527.5(ACDEFGHILMPQSTVWY), 528.5(DEPT), 529.5(ADEGLMNPTV), E530(P), 530.5(ACDEFGHILMNQRSTVWY), 531.5(ADEFGILMNPQSTVY), K532(R), 532.5(FHPSTVWY), F533(ACGHILMNQSTV), 533.5(FY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-CDNPWY), S537(-CDFGNQTY), G538(—), V539(AHLMNQS), 540.5(M), 541.5(D), F542(P), 542.5(Y), G543(APS), 543.5(ACDILPSY), K544(-ACFGHILMNPQSTVY), Q545(D), 546.5(E), S547(-DEIMQ), 547.5(CDV), 548.5(CDE), K549(CDFGILMPQTVWY), 549.5(DE), T550(CDE), 550.5(DE), N551(CDE), 551.5(CDEN), V552(AELMNST), I554(CEHPQSTW), 555.5(N), K556(ADEGMNQTV), V557(LM), M558(ADTV), 558.5(E), I559(C), 559.5(A), T560(AV), 560.5(A), D561(AFMQVY), E562(AD), R566(ACDEFGHILMNTVWY), T567(Q), 567.5(E), T568(P), 569.5(M), P570(QS), 570.5(M), V571(ACHLMQS), 571.5(G), A572(CGSY), T573(ACDGINSV), 573.5(MT), E574(DFLMQW), 574.5(V), Q575(DGIKLMNTV), 575.5(AFWY), Y576(ACGHIS), 576.5(EFWY), 577.5(ACHIMNQSTV), S578(DEFGHILNVWY), 578.5(AFGHLNST), V579(ACEFGHLMNQST), 579.5(FL), S580(-MNPQ), T581(CENP), N582(FILMTVWY), 584.5(NT), R585(K), 585.5(AFGILMPQSTVY), 586.5(AGPQS), N587(GILMQTV), 587.5(AIMNQV), 588.5(ILMV), Q589(APSTV), 589.5(AFPV), A590(MQV), 590.5(ACPQT), A591(CDHIT), 591.5(AFGILMNPTV), T592(-CDGMNP), 592.5(ACFGILMPSTY), A593(-DE), 593.5(AFGNPSTW), D594(E), 594.5(AGQSTW), V595(ACDEFGHILMNPQSTWY), 595.5(ADEFHILMQSTVW), N596(CDEFGMPTW), 596.5(DEN), T597(CDEGHIKLMPQRV), Q598(ACEFGHINSTVY), G599(C), L601(WY), P602(M), M604(ACS), V605(F), 605.5(N), 607.5(P), 610.5(F), F628(I), L633(FHMY), 640.5(N), S658(C), 658.5(C), T660(C), 662.5(W), N703(D), N705(DE), K706(EST), S707(D), V708(C), N709(DE), P723(E), R724(FHNTWY) .5(D), P30(D), E36(N), 38.5(K), 40.5(D), D41(AHQW), 41.5(D), S42(EGQ), 0.8-fold R43(T), 43.5(DHPSTV), 45.5(M), V46(I), G49(H), 50.5(N), K51(AY), G58(ET), K61(—), 61.5(K), E63(M), E74(H), 107.5(E), 135.5(M), 137.5(M), K142(I), 145.5(M), 150.5(M), 179.5(D), D180(M), 180.5(M), 183.5(M), 190.5(F), P192(M), 193.5(W), A194(C), H229(V), H255(N), L256(ACS), 260.5(ACDEMT), S261(D), S262(T), 262.5(DE), Q263(VW), 263.5(D), S264(DEFLW), 264.5(D), 265.5(DE), 266.5(E), S267(E), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(E), R307(DMN), P308(GM), T324(C), 324.5(C), Q325(C), 326.5(M), T330(Q), G355(AS), Q359(ADEHTV), F370(Y), T379(A), 379.5(DE), L380(EY), 380.5(E), N381(CD), N382(DE), 382.5(D), 383.5(E), 384.5(D), Q385(CGW), 385.5(DP), A386(E), 386.5(CDEP), V387(E), 387.5(EGP), G388(E), R389(ALMPSTY), S390(DT), 394.5(N), 404.5(N), G406(M), 408.5(C), 415.5(Q), 417.5(M), Y424(C), 424.5(F), Q428(EHS), S429(Y), D431(M), 431.5(Y), R432(I), L433(D), 433.5(F), L437(AHNY), I438(CGNT), D439(K), L442(FI), Y444(FH), 444.5(CLM), L445(CFMVY), 445.5(ACGHLMNQSTVY), S446(CDEGHILMNTV), 446.5(AFGHILMNPQSTVWY), R447(ACEFGHILMNQSTV), 447.5(CFGILMNPVWY), T448(CFGILMNPQVWY), 448.5(CEFILMPWY), N449(ACDEFGILMPSVW), 449.5(PY), T450(CDE), S452(DEFLV), 452.5(I), G453(L), 453.5(I), T454(DEFILM), 454.5(DEF), T455(DEL), T456(D), 456.5(DE), Q457(ADEILMV), 457.5(DEW), S458(CDEFMPVWY), 458.5(CDEFILMWY), R459(-ACDEFGHILMNPQSTVWY), L460(ACFHINPSTVWY), 460.5(W), Q461(CDEGKNTV), F462(Y), S463(ACDGNQV), 463.5(F), Q464(ACIKLMSTV), 464.5(S), A465(CGP), G466(ACDNQ), A467(CDFGHMPQY), S468(CEFLPQY), D469(ACEHMNQRSTV), 469.5(DE), I470(CFLM), R471(ACDEGKMQSTV), D472(Y), Q473(FHMSY), S474(I), R475(K), 475.5(SW), N476(STW), W477(Y), 477.5(L), L478(FI), P479(GIT), 479.5(D), G480(Y), P481(A), 481.5(HY), C482(FGQY), 482.5(FM), Y483(ACFHILMQTV), 483.5(FQ), R484(K), 484.5(N), Q485(ACFGHILNSTVY), 485.5(EKQY), Q486(FGINSVWY), 486.5(FV), R487(H), 487.5(FW), V488(ACEFGHLMNQTWY), 488.5(ACFGILMPQVWY), S489(-ACFGHIKLMPQTVY), 489.5(CDEPY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACFGHIMNPQSTV), T491(ADEM), 491.5(ACFILMPQTVWY), S492(CDEILMT), 492.5(ADEGILMPSV), A493(DEILPRV), 493.5(DIKLMPV), D494(R), 494.5(GIMPTV), N495(-AFILMPQSTVY), 495.5(AFGILMPSTVWY), N496(-ACFGHILMQSTVW), 496.5(FT), N497(-EITV), 497.5(ADEGILMQT), S498(-ADEFGILMPQRVWY), 498.5(DEFGLMPQY), E499(I), 499.5(ADEGHILMNPQRSTVWY), Y500(-ACDEGIKLMNPQST), 500.5(AEFGHILMNPQSTVW), S501(-ACEGILMNPTVWY), 501.5(ACFGHILMNPQRSTVWY), W502(-ACEGILMPQSVY), 502.5(AFGHILMNPQSTVWY), T503(-ADEFGIPSV), 503.5(AFGILMNPQSTVW), G504(-ACFILMNPQSTVWY), 504.5(ACFGHILMNPQSVWY), A505(-CEFGHILMNPQSTVWY), 505.5(ACFGILMPQSTVWY), T506(-ACFGILMNPQSV), 506.5(ACIPSTVW), K507(ACHILMNQSTVW), Y508(-DIMV), H509(CEFN), 509.5(K), L510(I), N511(ACDELMQWY), 512.5(S), R513(CHIV), D514(ACEGILMQSTV), S515(CIV), L516(ACIMSTV), V517(IL), 517.5(C), N518(CHILMQRTVY), P521(CFLMNST), 521.5(S), A522(CPV), M523(LV), 523.5(A), A524(CGPST), 524.5(ACHSTW), S525(ACGHMNP), 525.5(ACFGHILMNQSTVWY), H526(-ACDEFGIKLMNPQRSTVWY), 526.5(ACDEFGHILMNPQSTVY), K527(-HR), 527.5(ACDEFGHILMPQSTVWY), 528.5(DEPT), 529.5(ADEGLMNPTV), E530(P), 530.5(ACDEFGHIKLMNQRSTVWY), 531.5(ADEFGILMNPQSTVY), K532(-HRW), 532.5(FHIPSTVWY), F533(ACGHILMNQSTVW), 533.5(FHY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-CDFNPWY), 536.5(L), S537(-ACDFGNQTY), G538(—), V539(AHLMNQST), 540.5(KM), I541(DPY), 541.5(DGH), F542(P), 542.5(Y), G543(APS), 543.5(ACDHILPQSTVY), K544(- ACFGHILMNPQSTVY), 544.5(DE), Q545(-DEP), 545.5(W), G546(DE), 546.5(EILMT), S547(-CDEFGHILMNPQV), 547.5(ACDHNPVY), 548.5(CDEMP), K549(-ACDEFGHILMNPQSTVWY), 549.5(DE), T550(CDE), 550.5(DEG), N551(CDELY), 551.5(CDEN), V552(ACELMNST), I554(CEHPQSTW), 555.5(N), K556(ACDEGLMNQSTVWY), 556.5(Q), V557(LM), M558(ACDHINQSTVW), 558.5(E), I559(CV), 559.5(A), T560(AV), 560.5(A), D561(AEFGHILMNQTVY), E562(AD), E563(D), I565(T), R566(ACDEFGHILMNQSTVWY), T567(Q), 567.5(E), T568(PV), 569.5(M), P570(CQS), 570.5(M), V571(ACHLMQST), 571.5(G), A572(CGSY), T573(ACDGINSV), 573.5(MT), E574(CDFLMQW), 574.5(DV), Q575(DFGIKLMNTVW), 575.5(AFWY), Y576(ACGHIS), 576.5(EFWY), 577.5(ACHIMNQSTV), S578(DEFGHILNVWY), 578.5(AEFGHILNST), V579(ACEFGHLMNQST), 579.5(FLY), S580(-GMNPQ), T581(CEGKNP), N582(FILMTVWY), 583.5(Q), Q584(V), 584.5(FLNT), R585(K), 585.5(AFGILMPQSTVY), 586.5(AGMPQS), N587(GILMQTV), 587.5(AFIMNQV), 588.5(ILMV), Q589(APSTV), 589.5(AFPV), A590(GMPQV), 590.5(ACGPQST), A591(CDHIT), 591.5(AFGILMNPSTV), T592(-ACDGMNP), 592.5(ACFGILMNPSTY), A593(-DEN), 593.5(AFGNPSTW), D594(E), 594.5(AGHINQSTVWY), V595(ACDEFGHILMNPQSTWY), 595.5(ADEFHILMQSTVW), N596(CDEFGMPTW), 596.5(DEN), T597(CDEGHIKLMNPQRV), Q598(ACEFGHILMNSTVY), G599(CSW), L601(IWY), P602(M), M604(ACQS), V605(F), 605.5(N), 607.5(P), 610.5(F), K620(N), G626(A), F628(IM), P630(HIQ), L633(FHMY), 640.5(N), 657.5(C), S658(CE), 658.5(CQ), T660(C), 660.5(E), F661(LQ), S662(C), 662.5(FMW), A663(Y), 663.5(DM), 664.5(DE), A667(C), S674(E), 697.5(GN), N703(D), 703.5(E), N705(DE), K706(DEGST), S707(D), 707.5(Y), V708(CE), N709(DE), V710(E), 711.5(Y), T716(CE), N717(E), 720.5(N), S721(TV), E722(D), P723(CE), R724(FHNSTWY), 735.5(M) 3.5(DT), 4.5(N), Y6(I), D9(AV), R20(Q), K24(G), P27(T), P30(D), 32.5(L), 0.9-fold P34(L), 34.5(V), 35.5(IN), E36(N), R37(Q), H38(L), 38.5(FK), K39(-LPWY), 39.5(DP), D40(P), 40.5(DY), D41(AHKQTW), 41.5(DNV), S42(EGLQ), 42.5(AKNSTY), R43(AGPSTW), 43.5(DHPRSTV), 45.5(M), V46(IK), L47(F), P48(L), G49(HM), 50.5(N), K51(AMNY), P55(Y), 55.5(K), F56(V), G58(CEST), L59(AIQ), D60(N), K61(-Q), 61.5(K), G62(E), E63(CM), E67(DTV), A71(IT), L73(N), E74(DH), K77(Y), 78.5(Q), Q82(FM), 83.5(H), D84(I), D87(EQ), L91(W), K92(Y), A98(N), E99(K), Q101(IV), E102(QY), E106(H), D107(Q), 107.5(E), S109(N), F110(VY), R116(M), A117(N), A121(IL), L126(C), E127(F), L129(C), V132(AES), E133(—), 133.5(CFLPRSW), E134(-K), 134.5(W), 135.5(DEMW), 136.5(CNY), K137(L), 137.5(IM), 139.5(ADGT), P140(DHIT), 140.5(EN), G141(I), K142(CDEFHIT), 142.5(C), K143(—), P145(AY), 145.5(M), V146(NW), 146.5(DFNY), 147.5(DE), H148(DGNW), 148.5(E), 149.5(E), P150(DM), 150.5(DMP), V151(D), 151.5(WY), 152.5(M), S157(WY), K161(W), A162(H), T176(N), 177.5(IV), 178.5(D), 179.5(DN), D180(M), 180.5(M), S181(DF), 181.5(TWY), V182(W), P183(C), 183.5(LMY), P185(W), L188(Y), 188.5(EY), 189.5(Q), Q190(EW), 190.5(F), 191.5(W), P192(CMQW), 192.5(CDHM), A193(D), 193.5(DEPW), A194(CMW), P195(A), 195.5(TY), S196(FH), L198(C), N201(SV), 202.5(F), G206(E), G208(ET), A209(M), M211(V), N215(DI), G217(V), H229(DV), D231(C), S232(T), M235(V), T251(S), H255(N), L256(ACS), K258(LQ), 260.5(ACDEMQT), S261(DG), S262(T), 262.5(DEINT), Q263(EVW), 263.5(D), S264(DEFLMTW), 264.5(D), G265(—), 265.5(ADET), 266.5(AE), S267(ENT), 267.5(E), N268(ADEQ), 268.5(E), 269.5(D), N270(ACDGHQSY), H271(EIN), S276(T), H288(N), R307(-CDFMNS), 307.5(K), P308(-GMW), L311(I), N312(M), L315(I), T324(CL), 324.5(CL), Q325(CHIK), 325.5(I), 326.5(M), 327.5(P), G328(AHS), T329(CN), T330(Q), T331(L), I332(V), 332.5(K), L354(HT), G355(ACS), S356(N), Q359(ACDEGHNSTV), F370(Y), T379(ACV), 379.5(DE), L380(EY), 380.5(E), N381(CDGMQ), N382(DE), 382.5(D), 383.5(E), 384.5(D), Q385(CGSW), 385.5(ADEPTV), A386(CEFHNP), 386.5(CDEP), V387(DELM), 387.5(EGKP), G388(E), R389(ALMPQSTY), 389.5(N), S390(DT), 394.5(N), P399(EMQ), Q401(R), R404(K), 404.5(N), G406(M), 407.5(H), N408(HW), 408.5(C), F409(Y), T410(DHNY), S412(H), 412.5(Q), Y413(FW), T414(DK), 415.5(DQ), 416.5(EN), D417(NS), 417.5(KM), V418(IM), Y424(C), 424.5(F), A425(FGQ), S427(D), Q428(EHS), S429(CY), L430(I), D431(FM), 431.5(Y), R432(I), L433(ADMT), 433.5(F), L437(AHMNSY), I438(ACGHLNST), D439(K), L442(CFIV), Y443(F), Y444(FH), 444.5(CLM), L445(CFMVY), 445.5(ACGHILMNQSTVY), S446(ACDEGHILMNTV), 446.5 (AFGHILMNPQSTVWY), R447(ACEFGHIKLMNQSTV), 447.5(CFGILMNPVWY), T448(ACFGILMNPQVWY), 448.5(ACEFILMPWY), N449(ACDEFGILMPSTVW), 449.5(PY), T450(CDEI), S452(DEFLV), 452.5(IV), G453(EL), 453.5(I), T454(DEFILM), 454.5(DEFL), T455(ADEL), T456(DP), 456.5(DEGLP), Q457(ADEILMSTV), 457.5(DEPW), S458(ACDEFMNPTVWY), 458.5(CDEFILMPTWY), R459(-ACDEFGHILMNPQSTVWY), L460(ACFHIMNPSTVWY), 460.5(W), Q461(CDEFGKMNTV), F462(Y), S463(ACDGMNQTV), 463.5(F), Q464(ACIKLMSTV), 464.5(S), A465(CGP), G466(ACDNQ), 466.5(E), A467(CDFGHIMPQSVY), S468(CEFILNPQVY), D469(ACEHMNQRSTV), 469.5(DE), I470(CFLMV), R471(ACDEGKMQSTV), D472(Y), Q473(ACFHMSY), S474(CI), R475(K), 475.5(SW), N476(STW), W477(Y), 477.5(L), L478(FI), P479(GIT), 479.5(D), G480(Y), P481(AV), 481.5(HY), C482(FGIQVY), 482.5(FM), Y483(ACFHILMQTV), 483.5(FQ), R484(K), 484.5(N), Q485(ACFGHILNSTVY), 485.5(EKQY), Q486(FGIMNSTVWY), 486.5(FV), R487(-H), 487.5(FW), V488(ACEFGHILMNQTWY), 488.5(ACFGILMPQVWY), S489(-ACFGHIKLMPQTVY), 489.5(CDEPQY), K490(-ACDEFGHILMNPQRSTVWY), 490.5(ACFGHILMNPQSTVWY), T491(ADEILMPQSVW), 491.5(ACFGILMPQTVWY), S492(ACDEILMNQTV), 492.5(ADEGILMPQSV), A493(DEILPRV), 493.5(ADGIKLMPV), D494(R), 494.5(GILMPTV), N495(-AFGILMPQSTVY), 495.5(AFGILMNPSTVWY), N496(-ACFGHILMQSTVWY), 496.5(FLNT), N497(-EILTV), 497.5(ADEGILMQTV), S498(-ADEFGILMPQRVWY), 498.5(ADEFGLMPQY), E499(DI), 499.5(ADEGHILMNPQRSTVWY), Y500(-ACDEFGIKLMNPQSTV), 500.5(AEFGHILMNPQSTVWY), S501(-ACEFGILMNPTVWY), 501.5(ACFGHILMNPQRSTVWY), W502(-ACEFGILMPQSTVY), 502.5(AFGHILMNPQSTVWY), T503(-ADEFGIPQSVY), 503.5(AFGILMNPQSTVW), G504(-ACFHILMNPQSTVWY), 504.5(ACFGHILMNPQSVWY), A505(-CDEFGHILMNPQSTVWY), 505.5(ACFGILMPQSTVWY), T506(-ACFGILMNPQSV), 506.5(ACIPSTVW), K507(ACHILMNQRSTVWY), Y508(-DIMSTV), H509(CEFNQ), 509.5(K), L510(I), 510.5(K), N511(ACDEGLMQSWY), 512.5(S), R513(CHIV), D514(ACEGILMQSTV), S515(CITV), L516(ACIMSTV), V517(CIL), 517.5(C), N518(CHILMQRTVY), P521(CFLMNSTV), 521.5(MS), A522(CPSV), M523(LV), 523.5(A), A524(CGPST), 524.5(ACHSTW), S525(ACGHMNP), 525.5(ACFGHILMNPQSTVWY), H526(-ACDEFGIKLMNPQRSTVWY), 526.5(ACDEFGHILMNPQSTVWY), K527(-FHR), 527.5(ACDEFGHILMPQSTVWY), 528.5(DEPT), 529.5(ADEGLMNPTV), E530(DP), 530.5(ACDEFGHIKLMNQRSTVWY), E531(C), 531.5(ADEFGILMNPQSTVY), K532(-HRW), 532.5(FHIPSTVWY), F533(ACGHILMNQSTVW), 533.5(FHLY), F534(ILMVWY), 534.5(F), P535(-AF), Q536(-CDEFGNPTVWY), 536.5(L), S537(-ACDFGHNQTY), G538(—), V539(AHILMNQST), L540(M), 540.5(KMN), I541(-ACDGHPQTY), 541.5(DEGHMQST), F542(KP), 542.5(Y), G543(APS), 543.5(ACDHILMPQSTVY), K544(-ACFGHILMNPQRSTVY), 544.5(ADEV), Q545(-DEFHLPVY), 545.5(CDNPTW), G546(ACDEMNPS), 546.5(CDEGILMPQSTV), S547(-CDEFGHILMNPQVY), 547.5(ACDFGHLNPQSTVWY), 548.5(CDELMPW), K549(-ACDEFGHILMNPQSTVWY), 549.5(ADELQTV), T550(ACDEGLNQ), 550.5(ADEGPST), N551(CDEFLMQSY), 551.5(CDENV), V552(ACEILMNST), 552.5(V), I554(ACEFHLMPQSTVW), 555.5(N), K556(ACDEGLMNQSTVWY), 556.5(Q), V557(ILM), M558(ACDHINQSTVWY), 558.5(E), I559(CFMQV), 559.5(A), T560(ACSV), 560.5(A), D561(AEFGHILMNQSTVY), E562(AD), E563(DG), I565(T), 565.5(Y), R566(ACDEFGHILMNQSTVWY), T567(AGPQS), 567.5(E), T568(PV), 569.5(M), P570(CQS), 570.5(M), V571(ACHLMQST), 571.5(G), A572(CGSY), 572.5(V), T573(ACDGINSVW), 573.5(MT), E574(CDFLMQW), 574.5(DV), Q575(-DFGIKLMNTVWY), 575.5(AFWY), Y576(ACFGHIS), 576.5(EFWY), 577.5(ACHIMNQSTV), S578(ADEFGHILNVWY), 578.5(ACEFGHILNST), V579(ACDEFGHILMNQST), 579.5(FGILMY), S580(-GMNPQT), T581(CEGKNP), N582(FILMSTVWY), 583.5(Q), Q584(AV), 584.5(FLNQT), R585(K), 585.5(AFGILMPQSTVY), G586(LMQ), 586.5(AGMPQST), N587(GILMQTV), 587.5(AFILMNQV), 588.5(ILMV), Q589(APSTV), 589.5(AFPV), A590(GILMNPQV), 590.5(ACGPQST), A591(CDHILMT), 591.5(AFGILMNPSTV), T592(-ACDEGMNPS), 592.5(ACFGILMNPSTVY), A593(-DEFNPY), 593.5(AFGMNPQSTVW), D594(E), 594.5(AFGHINQSTVWY), V595(ACDEFGHILMNPQSTWY), 595.5(ADEFHILMNQSTVW), N596(ACDEFGMPQTW), 596.5(DEN), T597(CDEGHIKLMNPQRVY), Q598(ACEFGHILMNSTVY), G599(CSW), V600(C), L601(FIMVWY), P602(M), M604(ACQS), V605(F), 605.5(N), 607.5(P), 610.5(F), Y612(F), Q614(N), P616(Q), A619(C), K620(ACMN), I621(AKL), H623(P), T624(V), D625(S), G626(A), F628(HIM), P630(CGHIQTV), 630.5(CHV), L633(FHMY), M634(C), 640.5(N), H641(M), Q645(LM), 655.5(IW), N656(FQ), 657.5(CH), S658(CEHMQ), 658.5(CEFGMQ), T659(CDHMS), 659.5(DEHPQY), T660(CDM), 660.5(DEF), F661(ELQTW), 661.5(LPS), S662(CDGP), 662.5(CDEFHMPW), A663(DEFNY), 663.5(CDEMW), A664(DEG), 664.5(ADEGNS), K665(P), 665.5(NP), F666(H), 666.5(V), A667(C), 667.5(Q), F669(Y), S674(E), E681(M), E697(M), 697.5(GNPW), I698(V), 699.5(E), Y700(F), 701.5(A), S702(N), N703(D), 703.5(E), N705(DEM), 705.5(E), K706(DEGST), S707(D), 707.5(Y), V708(CE), N709(CDE), V710(E), 711.5(QY), T716(CEM), N717(DE), V719(C), 720.5(N), S721(CIMQTV), E722(D), P723(ACEHIQTV), R724(FHNSTWY), P725(L), L735(IV), 735.5(LMV)

The preceeding describes a wide range of individual amino acid mutations that are tolerated and affect viral tropism, positively or negatively. It is specifically comtemplated herein that combinations of these various mutations can be made to further alter viral tropism, or other properties. In one embodiment, for example, at least 2 of the single amino acid mutations described herein are introduced in combination. For example, 2 mutations that influence tropism for liver can be combined. It is anticipated that the resulting virus will also have increased tropism for liver. In further embodiments, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations described herein are introduced in combination. We have demonstrated herein that a wide range of mutations are tolerated, however, no more than 40% of the amino acids should change relative to a reference sequence (e.g., SEQ ID NO: 1). In certain embodiments, no more than 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of amino acids, or fewer, are changed relative to a reference sequence (e.g., SEQ ID NO: 1).

Mutations that are at equivalent positions in other homologous viruses can be made and used for improving virus packaging and virus infectivity. Examples of other homologous viruses include any of AAV serotypes 1, and 3-12, as well as other natural isolates or synthetic sequences. Corresponding positions in homologous viruses can be inferred from sequence homology to AAV2. In one embodiment, the mutations described herein are introduced into the corresponding amino acid sequence of an AAV1, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13 capsid polypeptide.

Site-directed mutagenesis is known in the art and can be used to introduce a point mutation(s) (e.g., amino acid substitutions, insertions, or deletations) or other mutations or combinations thereof to a viral capsid polypeptide. Site-directed mutatgenesis is further described in, e.g., Li B, et al. Hum Gene Ther Methods. 2015 December: 26(6):211-20, and Bachman, J. Methods Enzymol. 2013; 529:241-248, which are incorporated herein by reference in their entirety.

Combinations of mutations can be used to achieve a particular vector targeting goal. For example, it may be desirable to use a virus that has improved nucleic acid delivery in a first tissue and reduced nucleic acid delivery in one or more second tissues. Such a virus, whether achieved through a single mutation or multiple mutations, can have increased tissue specificity.

Mutatable Subregions of AAV2 Capsid Protein

It was found herein that particular regions of AAV2 capsid protein tolerate amino acid alterations, e.g., deletion, insertion, or substitution better than others, and that changes to certain regions that tolerate such change have pronounced impact on viral vector tropism. The region of the AAV2 capsid polypeptide of SEQ ID NO: 1 from amino acids 440 to 600 is identified herein as both tolerant of a variety of changes and highly influential for viral tropism, with certain mutations influencing tropism towards or away from certain tissues or cell types, as indicated in the Tables herein. Within this region, sub-regions including amino acids 440-460, 475-505, 518-532 and 560-590 are seen, for example, in the heat maps provided herein, to be particularly important for changes in viral tropism, and one of these sub-regions, from amino acids 561-588 is shown herein to tolerate extensive mutation. This region, which was found in single amino acid mutation studies to be important for tropism, was selected for initial studies of the effect of combinations of mutations. It was found that combinations of two to at least 8 different single amino acid mutations within this region were well tolerated, and further influenced tropism, as shown in the SEQ ID Nos: 3-20.

SEQ ID NO: 2 is an amino acid sequence encoding the amino acid region between amino acids 440-600 of SEQ ID NO: 1.

(SEQ ID NO: 2) QYLYYLSRTNTPSGITTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSK TSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLI FGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAAT ADVNTQGV

TABLE 10 Amino acid alterations between amino acids 440-600 of SEQ ID NO: 1 conferring more efficient delivery to blood. Fold increase of Amino acid alteration efficiency 445.5(D), S446(—), 446.5(DE), R447(-PWY), 447.5(DEHQ), T448(-DEH), 1.1-fold 448.5(D), N449(H), T450(FS), 452.5(P), G453(V), T454(APQ), T455(GMS), T456(A), 456.5(A), 457.5(MT), Q464(R), 464.5(Y), S468(M), C482(MSW), Y483(W), R484(ILQV), 485.5(M), Q486(DE), R487(ACDEFGILMNPQSTVWY), 488.5(DE), S489(DE), 490.5(DE), T491(FN), 491.5(DE), A493(FS), 493.5(ENST), 494.5(E), N495(DEW), 495.5(DE), N496(DE), 496.5(DE), N497(AMP), 498.5(ST), 500.5(D), S501(D), 501.5(DE), 502.5(DE), 503.5(DE), G504(DE), 504.5(E), 505.5(E), K507(-EF), P521(I), M523(C), S525(T), 525.5(DE), K527(PQW), D528(Q), E531(DGMN), K532(ADEFGILMNPQSTVY), 532.5(DEMN), F533(DEY), 533.5(CDEGSV), 536.5(V), V539(C), V571(DE), Q575(A), Y576(M), 577.5(E), V579(VV), 579.5(CDEN), S580(ADE), 580.5(DEILMPY), T581(DSWY), 581.5(DEFILMPQV), N582(-ACDEPQ), 582.5(GP), L583(-DEGPT), 583.5(DEIPV), Q584(-DEFLNTY), 584.5(ADEIMVY), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-DEP), 586.5(DEFHINVY), N587(-DE), 587.5(DEGPY), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEFQTY), Q589(DEFMNY), 589.5(DEGINS), A590(DEW), 590.5(DEFY), A591(EFPQSY), 591.5(CDEQWY), T592(FY), 592.5(DEQ), A593(GLMTVW), 593.5(CDEILY), 594.5(CDEP), 595.5(C), N596(S), T597(AS), V600(AILST) 445.5(D), S446(—), 446.5(DE), R447(-P), 447.5(DEHQ), T448(-DE), 1.5-fold N449(H), T454(P), 464.5(Y), C482(M), R484(ILQV), 485.5(M), Q486(DE), R487(ACDEFGILMNPQSTVWY), 488.5(E), S489(DE), 490.5(DE), T491(N), N495(DEW), 495.5(DE), N496(DE), 496.5(DE), S501(D), 501.5(D), 502.5(DE), 503.5(DE), G504(DE), K507(-E), 525.5(D), E531(GMN), K532(ADEFGILMNPSTVY), 532.5(DEM), F533(DE), 533.5(CDEGSV), V571(DE), 577.5(E), V579(V), 579.5(CDE), S580(DE), 580.5(DEILMY), T581(W), 581.5(DEFILMPQV), N582(-ACDEP), 582.5(GP), L583(-DEPT), 583.5(DEIPV), Q584(-DEFLTY), 584.5(DEIV), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-DE), 586.5(DEFHY), N587(-DE), 587.5(DEP), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEFY), Q589(DEFY), 589.5(DEIN), A590(DEW), 590.5(DE), A591(PY), 591.5(CDEY), T592(FY), 592.5(DE), 593.5(CDE), 594.5(CDE), 595.5(C), N596(S) 445.5(D), S446(—), 446.5(DE), R447(-P), 447.5(EHQ), T448(D), T454(P), 2-fold 464.5(Y), R484(ILQV), 485.5(M), Q486(DE), R487(ACDEFGILMNPQSTVWY), 488.5(E), S489(DE), 490.5(D), N495(DEW), 495.5(DE), N496(DE), 496.5(DE), S501(D), 501.5(D), 502.5(E), 503.5(DE), G504(DE), K507(—), K532(ADEGILMNPSTV), 532.5(DE), F533(DE), 533.5(CDEGSV), V571(DE), 577.5(E), 579.5(DE), S580(DE), 580.5(DEILMY), T581(W), 581.5(DEFILMPQV), N582(-DEP), 582.5(GP), L583(-DEP), 583.5(DEIPV), Q584(-DEFY), 584.5(DEIV), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-DE), 586.5(DEY), N587(-DE), 587.5(DEP), R588(-ACDEFGHILMNPQSTVWY), 588.5(DE), Q589(DE), 589.5(DEIN), A590(DEW), 590.5(DE), A591(PY), 591.5(DE), T592(FY), 592.5(DE), 593.5(DE), and 594.5(CDE) N496(DE), S501(D), G504(D), K532(DE), 581.5(DEIPV), N582(DE), 5-fold 582.5(G), L583(-DE), 583.5(DEP), Q584(-DE), 584.5(DE), R585(-ADEFGHILMNPQSTVWY), 585.5(DE), G586(-D), 586.5(DE), N587(-DE), 587.5(DEP), R588(-ACDEFGHILMNPQSTVY), 588.5(DE), 589.5(DE), A590(DE), 590.5(D), 593.5(E), and 594.5(E) R585(D) 10-fold

TABLE 11 Amino acid alterations between amino acids 440-600 of SEQ ID NO: 1 conferring more efficient delivery to heart. Fold increase of Amino acid alteration efficiency Y444(F), 448.5(S), N449(A), T450(M), S452(M), T454(LQ), 455.5(A), 1.1-fold T456(AV), Q457(DLV), S458(AP), 458.5(Q), R459(FH), Q464(AI), A467(CGNPQ), S468(ADG), D469(AENQST), I470(LMV), R471(CKM), C482(M), Q486(N), R487(ACGIMQSTV), S489(AD), 490.5(N), T491(FLW), 492.5(M), A493(M), N496(DHSY), N497(PV), S498(FM), Y500(M), T506(S), V517(I), S525(AG), H526(CNST), E531(D), K532(ADEFGILMNQRST), I541(C), K544(CGTVY), Q545(E), K549(Q), N551(Y), V552(Q), K556(DNY), D561(Q), R566(ASV), T567(S), S578(TV), V579(V), S580(A), T581(WY), 581.5(IV), N582(DE), L583(DE), 584.5(EIV), R585(-ADFGHILMNQSTVWY), G586(DE), N587(DE), 587.5(D), R588(ACDEFHILMNQSTVY), 588.5(DE), Q589(M), A590(P), A591(ETY), 591.5(CDE), T592(F), 594.5(D), T597(ANSW), Q598(L), V600(AS) D469(AQS), I470(LM), R471(K), R487(A), S489(D), 490.5(N), T491(W), 1.5-fold N496(D), S498(M), K532(ADEL), D561(Q), N582(DE), L583(E), 584.5(V), R585(-AGHIMNQSTVWY), N587(DE), R588(ADEFILMNQSTVY), 588.5(DE), Q589(M), 591.5(D), T597(W), and V600(A) R471(K), K532(D), N582(D), L583(E), R585(Q), N587(DE), R588(MQTV), 2-fold and 588.5(E)

TABLE 12 Amino acid alterations between amino acids 440-600 of SEQ ID NO: 1 conferring more efficient delivery to kidney. Fold increase of Amino acid alteration efficiency 446.5(D), 447.5(HQ), N449(ACGS), T456(AG), 456.5(G), R459(HK), 1.1-fold Q464(A), A467(GNP), S468(AG), D469(AEQST), I470(LMV), R471(K), C482(M), Q486(D), R487(P), S489(E), 490.5(N), T491(F), A493(FI), N495(E), 495.5(DE), N496(EHS), 496.5(D), N497(P), S498(M), Y500(M), S501(D), 501.5(D), G504(D), N518(S), H526(CS), K532(DER), 532.5(D), F533(Y), K544(ILTV), T550(G), K556(N), D561(Q), R566(AV), S578(TV), V579(V), S580(A), 580.5(IMY), 581.5(EFILMPQV), N582(-ACDEP), 582.5(GP), L583(-DEIP), 583.5(DEIPV), Q584(-EFY), 584.5(DEILV), R585(-DEINPQVW), 585.5(D), G586(—), 586.5(DEIY), N587(-D), 587.5(DEP), R588(-ACDEGHLNPQTV), 588.5(DE), Q589(MP), 589.5(DE), A590(DEPV), 590.5(DE), A591(PST), 591.5(C), A593(M), 593.5(CE), T597(ASW), Q598(L), V600(A) D469(AQ), I470(L), R471(K), 490.5(N), 495.5(DE), N496(EHS), S498(M), 1.5-fold S501(D), K532(D), 580.5(I), 581.5(EILMPV), N582(-D), 582.5(GP), L583(DE), 583.5(DEIPV), Q584(-EFY), 584.5(IL), R585(-IP), G586(—), N587(—), 587.5(P), R588(-CDGV), 588.5(D), 589.5(DE), A591(P), 591.5(C), 593.5(C), T597(W), and V600(A) R471(K), K532(D), 581.5(IM), L583(DE), 583.5(D), 584.5(L), G586(—), 2-fold N587(—), 587.5(P), R588(—), 591.5(C), and 593.5(C)

TABLE 13 Amino acid alterations between amino acids 440-600 of SEQ ID NO: 1 conferring more efficient delivery to liver. Fold increase of Amino acid alteration efficiency Y444(F), N449(S), T450(IV), S452(A), 455.5(A), T456(V), Q457(T), 1.1-fold S458(AP), R459(HKT), L460(CN), A467(CGPQ), S468(G), D469(AENQST), I470(LMV), R471(CKM), C482(IM), Y483(M), Q486(N), 490.5(N), T491(DE), A493(MP), N496(HS), S498(AFLM), Y500(M), T503(V), T506(ACV), K507(S), V517(I), S525(G), H526(ACNST), K532(HINQWY), F533(Y), K544(C), 545.5(D), T550(E), I554(L), K556(ENY), M558(F), D561(Q), E563(D), R566(ACGNSTV), T567(S), S578(DEIT), V579(V), S580(A), T581(DM), L583(I), A590(IP), A591(DEQS), T592(S), A593(DET), N596(C), T597(ACDHLNQW), Q598(ILMV), V600(AST) R459(H), L460(N), A467(CGPQ), D469(AEQST), I470(LM), R471(K), 1.5-fold C482(I), Q486(N), N496(H), S498(M), H526(CNS), K532(N), K544(C), T550(E), K556(Y), R566(AGTV), A590(P), A591(E), A593(DE), T597(HNQ) A467(G), D469(AEQS), I470(L), R471(K), Q486(N), N496(H), S498(M), 2-fold H526(S), T550(E), A593(DE), and T597(N)

TABLE 14 Amino acid alterations between amino acids 440-600 of SEQ ID NO: 1 conferring more efficient delivery to lung. Fold increase of Amino acid alteration efficiency Y444(F), N449(Q), T450(I), S452(M), T454(IQ), T455(G), S458(AP), 1.1-fold 458.5(Q), R459(F), Q461(G), A467(CGNPQ), S468(G), D469(AENQST), I470(LMV), R471(K), 490.5(N), T491(QW), N497(P), N518(M), P521(V), F533(Y), V539(C), K544(C), Q545(N), T550(A), K556(N), D561(Q), S578(T), S580(A), Q589(M), A590(P), A593(T), T597(AS), V600(A), S264(G), A467(G), D469(AQT), I470(M), R471(K), 490.5(N), and V600(A) 1.5-fold

TABLE 15 Amino acid alterations between amino acids 440-600 of SEQ ID NO: 1 conferring more efficient delivery to spleen. Fold increase of Amino acid alteration efficiency L445(I), S458(A), R459(K), S468(G), D469(Q), T491(LQ), Y500(W), 1.1-fold F533(Y), I541(C), M558(F), V579(V), S580(A), Q589(MN), A590(P), A591(S), A593(MT), T597(AMY), V600(AIT) Q589(M), and V600(A) 1.5-fold

Interestingly, when combinations of mutations were examined, it was found that if a single mutation identified to be beneficial, e.g., for a change in tropism, is introduced, the resulting polypeptide tends to tolerate further mutation to include other single amino acid changes found to be beneficial. This is illustrated in, e.g., FIG. 3, which shows fitness at first mutation versus the number of mutations away from wild-type that will be tolerated before the virus is no longer viable. On the x-axis is the fitness at first mutation, i.e., whether a single mutation is neutral, deleterious, or beneficial. On the y-axis, is the number of mutations tolerated before the virus is no longer viable. The figure shows that if a virus has a first mutation that is beneficial, it will tolerate significantly more mutations, as compared to a virus with a first mutation that is neutral or deleterious. Thus, it is anticipated that combinations of mutations that do not interfere with viral fitness will be well tolerated.

Further identified herein are sub-regions (e.g., regions within the full length polypeptide) within the viral capsid polypeptide that are more likely to tolerate an amino acid change. One aspect herein is an AAV2 capsid polypeptide comprising a mutation in the sub-region of amino acids selected the group consisting of: 440-460 of SEQ ID NO: 1, 475-505 of SEQ ID NO: 1, 518-532 of SEQ ID NO: 1, and 560-590 of SEQ ID NO: 1 that alters tissue tropism of a virus comprising the viral capsid polypeptide. In further embodiments, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations described herein are introduced in combination into a sub-region. Though a wide range of mutations are tolerated, no more than 40% of the amino acids within a sub-region should change relative to a reference sequence (e.g., SEQ ID NO: 1). In certain embodiments, no more than 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of amino acids within a sub-region, or fewer, are changed relative to a reference sequence (e.g., SEQ ID NO: 1).

In one embodiment, a mutation described herein more efficiently increases viral tropism when introduced into a particular sub-region of the viral capsid polypeptide. In one embodiment, delivery is at least 1.1-fold more efficient as compared to a wild-type viral capsid polypeptide. In another embodiment, the delivery of a nucleic acid is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 5-fold, at least 10-fold, or more, more efficient as compared to a virus comprising the wildtype SEQ ID NO: 1 capsid polypeptide.

One aspect of the technology is a method of altering tropism of a virus comprising modifying a capsid polypeptide corresponding to the polypeptide of SEQ ID NO: 1 to introduce a mutation in at least 2 regions selected from the group consisting of: amino acids 440-460 of SEQ ID NO: 1, amino acids 475-505 of SEQ ID NO: 1, amino acids 518-532 of SEQ ID NO: 1, and amino acids 560-590 of SEQ ID NO: 1.

One aspect is an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the kidney, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of:

(SEQ ID NO: 3) DEEEIATTNPVATEQYGDVSENLMHFQN; (SEQ ID NO: 4) DEEEIRQTNPVATEGYGEVSTNLMHGNK; (SEQ ID NO: 5) DEEEIRTTNPVATEQYGIVnStTNLNEGNR; (SEQ ID NO: 6) DEEEIRTTNPVATECYGSVSTDLQSGNL; (SEQ ID NO: 7) DENEIRTTNPVATEIYGSVSTeNLQNnGdNR; (SEQ ID NO: 8) DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR; (SEQ ID NO: 9) DEEEIRTTNPVATEQYGDVSENLMHFQN.

An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the liver, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of:

(SEQ ID NO: 10) DEEEIRTTNPVATEQYGVVSDNLQRGNR; (SEQ ID NO: 11) DECEIRTTNPVATEQYGSVGENLQRGNR; (SEQ ID NO: 12) DEEEIRTTNPVATEQYGVVSENLQRGNR; (SEQ ID NO: 13) DESEITTTNPVATEQYGWVSTNQQRGNR; (SEQ ID NO: 14) HELEIATTNPVATEQYGSASTNIQRGNR; (SEQ ID NO: 15) DEEEIATTNPVATEQYGGVSTNLQRGNR.

An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the lung, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of:

(SEQ ID NO: 16) DEEEIVTTNPVATEQYGNVSTNLQRGNR; (SEQ ID NO: 17) DEDEISTTNPVATEQYGSCSTNLQRGNR; (SEQ ID NO: 18) QEEEIRTTNPVATEQYGSVSTNLQRGDR; (SEQ ID NO: 19) NEEEIRTTNPCATEVYGSVSTNLQRGNR; (SEQ ID NO: 20) DEQEIVTTNPVATEVYGTVSTNLQRGNR.

One aspect of the technology described herein is a nucleic acid encoding any of the viral capsid polypeptides described herein.

Another aspect of the technology described herein is a viral particle comprising any of the viral capsid polypeptides described herein.

Corresponding Mutations

Mutations at corresponding locations in, for example homolgous viral capsid polypeptides, would be expected to have similar effects on viral tropism. Corresponding locations can include, for example, a location relative to the full length capsid polypeptide (e.g., SEQ ID NO: 1), or for that matter, a location relative to a sub-domain or sub-region of the the full length capsid polypeptide (e.g., SEQ ID NO: 2). In one aspect, a viral capsid polypeptide is provided herein that bears a mutation that corresponds to a mutation of the polypeptide of SEQ ID NO: 1 as described herein or selected from the group consisting of the mutations in Tables 1-9 relative to SEQ ID NO: 1. In one embodiment, a homologous viral capsid polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to wild-type AAV2 capsid protein (e.g., SEQ ID NO: 1). In another embodiment, the viral capsid polypeptides of AAV serotypes 1, and 3-13 are homolgous to a AVV2 capsid polypeptide.

One aspect described herein is a viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO: 2, wherein the region corresponding to the amino acid sequence of SEQ ID NO: 2 comprises a mutation relative to SEQ ID NO: 2 that alters tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 10-15. In one embodiment, a homologous viral capsid polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 2.

As noted elsewhere herein, the identification of amino acid sites or regions of amino acid sequence in the AAV2 capsid polypeptide of SEQ ID NO: 1 that tolerate change in general, i.e., that permit packaging and infection by viral particles, as well as regions that influence tropism and/or packaging, either positively or negatively, can be used to guide changes in the capsid polypeptides of other AAV serotypes to similarly influence function or provide for modified properties. Thus, through use of sequence alignment, a mutation or set of mutations that provides a desired change in tropism and/or packaging for the AAV2 capsid polypeptide of SEQ ID NO: 1 can be introduced to the corresponding location of the capsid polypeptide of another AAV serotype to similarly influence those properties in that serotype. As but one example, it was determined herein that the region from amino acids 440 to 600 of the AAV2 capsid polypeptide of SEQ ID NO: 1 is important in determining viral tropism, and indeed, that there are sub-regions within that region, e.g., amino acids 561-588, that robustly influence tropism. It is contemplated that the introduction of changes identified herein in this amino acid 440 to 600 region, or in the amino acid 561-588 sub-region of the AAV2 capsid polypeptide to the corresponding region or sub-region of another AAV serotype capsid polypeptide will influence tropism of that AAV serotype in a similar manner.

Viral Packaging

One embodiment of the technology described herein relates to a method of increasing viral packaging by modification of an AAV2 capsid polypeptide to comprise at least one mutation in the region of amino acids selected the group consisting of: 34-38 of SEQ ID NO: 1, 133-152 of SEQ ID NO: 1, 188-194 of SEQ ID NO: 1, and 654-662 of SEQ ID NO: 1.

One embodiment of the technology described herein relates to a method of increasing viral packaging efficiency by modification of an AAV2 capsid polypeptide comprising any non-positive amino acid insertion, substitution, or deletion in the region of amino acids selected from the group consisting of: 442-452 of SEQ ID NO: 1, 489-506 of SEQ ID NO: 1, 541-551 of SEQ ID NO: 1, and 577-596 of SEQ ID NO: 1. In another embodiment, packaging efficiency is increased by the mutation of a plurality of amino acids to any non-positive amino acid insertion, substitution, or deletion in the region of amino acids selected from the group consisting of: 442-452 of SEQ ID NO: 1, 489-506 of SEQ ID NO: 1, 541-551 of SEQ ID NO: 1, and 577-596 of SEQ ID NO: 1.

TABLE 16 Amino acid alterations conferring more efficient viral packaging. Fold increase of Amino acid alteration efficiency D13(N), T14(HQ), I19(AV), Q21(F), W22(Y), W23(EFHIKLNQTVY), 1.1-fold K24(M), L25(CI), K26(H), 28.5(AV), P29(ADFGIVY), P31(HIKNQT), 32.5(Q), K33(AFILNPQRST), P34(A), 34.5(AY), A35(-E), 35.5(CKPQSTVW), E36(GV), 36.5(ADGLV), R37(G), 37.5(ADEGNPQRV), H38(ADEGKPQVY), 38.5(ADE), K39(A), R43(T), P48(Y), E63(DHIP), V65(NS), N66(D), E67(A), A70(KQ), L73(N), K77(E), A78(K), D80(EHNQ), R81(H), D84(ET), S85(QT), N88(FV), K92(ACFLT), A98(Q), E99(DKQTV), Q101(HN), E102(Y), R103(AT), K105(DHN), E106(DTW), 108.5(H), S109(A), F110(K), L114(F), G115(A), R116(AFGIKLQST), A117(FLQSTW), V118(CFGILSTWY), Q120(EN), K123(C), R124(ADEHKNQT), V125(CFGILT), L126(ACDFGHNSTVY), E127(ADFGHIMNPSTY), L129(AFGHNPSWY), 129.5(P), G130(DHQ), L131(ACFGHQRWY), V132(CHY), 132.5(DE), 133.5(EKNT), E134(AHIKLNPQSTV), 134.5(ADEGHIKLNPQRSTVY), P135(-ACDEFGHKNQRSTVWY), 135.5(CEFKMY), V136(ACDEFGHIKNPQRSTWY), 136.5(ADEFGNPSTW), K137(ET), 137.5(AKT), T138(-DHY), 138.5(ADEGINPQSTWY), A139(-DEGNPVW), 139.5(ACDEFGHILNPQSTVWY), P140(-ADEFNQSTVW), 140.5(ACDEFGHILNPSTVWY), G141(-ACDEFLNPQSTVWY), 141.5(ADFGHILPQSTVY), K142(-AEFGHILNQRSTVY), 142.5(FT), K143(—), P145(CFHLQT), 145.5(ACFGHILNPSVWY), V146(FGINPQWY), 146.5(DEGINQTVY), E147(DI), 147.5(ACDEFGHILNPQSTVY), H148(-ACDEFGLNPQSTVWY), 148.5(CDEGLNPQSTVY), 149.5(PS), P150(FINWY), 150.5(ACEFGHIKLPQRSTVWY), V151(IQY), 151.5(ACDEHILPQRVWY), E152(LSWY), 152.5(F), S157(TWY), T159(FILQVY), K161(CF), A162(CIKNQSTVW), Q164(ACDFHKLNPRVWY), P166(H), Q175(WY), 175.5(D), 177.5(CFW), A179(P), D180(W), 180.5(S), S181(DEQ), 181.5(CST), V182(CIW), Q186(IVWY), 188.5(DST), G189(DENY), 189.5(CDEFGHINSTY), Q190(-CDENSTW), 190.5(ADEFGIKLNPQSTVW), P191(-CDEFGHIKLMNQSTVWY), 191.5(ADEFGHILMNPQSTVY), P192(-ADEFQRSTY), 192.5(AGKQST), A193(EFKP), 193.5(AEP), A194(ST), 194.5(EY), P195(-D), S196(A), T200(CY), T202(CW), T205(AH), A209(S), E216(D), 237.5(D), 262.5(D), Q263(G), S267(T), N270(CDS), Y275(W), S276(A), R310(K), T324(S), 324.5(R), T344(AEHS), S346(DEKN), L354(IV), H358(ACQS), Q359(DE), L362(F), F370(Y), M371(LTV), V372(I), G376(A), L380(V), N381(C), N382(D), Q385(W), V387(L), P399(A), N408(T), T410(N), T414(Y), V418(M), Y424(F), A425(C), L433(TV), I438(ACLNTV), L442(F), Y443(F), Y444(F), L445(CMV), 445.5(ACDIMNQSTV), S446(-ACEHMNQTV), 446.5(ACDEFILMNPQSTVW), R447(-ACDEFGHILMNPQSTVWY), 447.5(CDEFGHILMNPQVWY), T448(-DEFHILMNPQVWY), 448.5(ADEFLMPY), N449(ACDEFGILMPQSVW), T450(EIS), 450.5(E), S452(DFL), 452.5(P), G453(EL), T454(EIM), 454.5(DE), T455(ADE), T456(D), 456.5(DG), Q457(ADELSV), 457.5(DE), S458(DENPTW), 458.5(DEFMWY), R459(-ACDEFGHILMNPQSTV), L460(ACIMNPSTV), Q461(E), S463(N), Q464(AIV), A467(GMNPS), S468(AGHMQ), D469(AEST), I470(CLMV), R471(ACKMQST), D472(Y), Q473(FH), C482(M), Y483(CIMVW), R484(IKLQV), 484.5(N), Q485(ACFIV), Q486(DEGNSTV), R487(ACDEFGHILMNPQSTVWY), V488(ACFILMWY), 488.5(DEP), S489(ADEGHMPQW), K490(-ACDEFGHILMNPQRSTVWY), 490.5(DFGIMNQTVWY), T491(ADEFGMQSWY), 491.5(ADEFILNPQTV), S492(DEHMT), 492.5(ADEGILMQV), A493(DEFILMV), 493.5(DEINSV), 494.5(DEM), N495(ADEFILMPQVWY), 495.5(DEFGWY), N496(ACDEFGHILMQSVWY), 496.5(DE), N497(-AEILMTV), 497.5(DEGIMQST), S498(-ADEFGLMQTWY), 498.5(ADFGLMQS), E499(D), 499.5(ADELMNPQSTV), Y500(-ADEGLMNPQSTVW), 500.5(ADEFGLMNPQST), S501(-CDEFGILMNPQTV), 501.5(ACDEGHILMNQSTVWY), W502(P), 502.5(ADEFGHILMNPQSTVWY), T503(AFIPVY), 503.5(ADEFILMNSTVW), G504(ACDEFILMNPVWY), 504.5(ACEFILMQSTVWY), A505(EGLSY), 505.5(CEFILMV), T506(ACILMNPV), 506.5(ACIV), K507(-CFHILMNQSTVWY), 510.5(K), N511(ACDM), R513(V), S515(TV), L516(ACIMSTV), V517(I), N518(QT), P521(ILT), A522(P), M523(CQ), A524(G), 524.5(T), S525(CG), H526(-ACDEGILMNPQRSTVW), K527(FHR), E531(DM), K532(-ADEFGHILMNPQRSTVWY), 532.5(AE), F533(ACDEGHILMNQSTV), 533.5(CDEGS), F534(MW), P535(—), Q536(-CDEFPTVY), 536.5(CV), S537(-ACDFGNQTY), G538(—), V539(CMST), I541(CH), K544(AILMQT), 544.5(D), Q545(EM), 545.5(E), G546(H), 546.5(D), K549(CNQT), 549.5(E), T550(AD), N551(DEMY), I554(QV), K556(DE), M558(N), D561(Q), R566(ACGMNSTVW), V571(ACES), A572(G), T573(CG), E574(D), Q575(GILMNTV), Y576(CM), 577.5(E), S578(DEGILNWY), 578.5(A), V579(ACEGIMNSTWY), 579.5(DEN), S580(-DEGINPQT), 580.5(DEL), T581(-DEMNPSWY), 581.5(DEILPQ), N582(-ACDEFILMPQSTVWY), 582.5(GP), L583(DEGIPTV), 583.5(DEPQV), Q584(DEGILTV), 584.5(ADEFGILMQSTVY), R585(-ADEFGHIKLMNPQSTVWY), 585.5(ADEFGILMNPQSTVY), G586(-DELMP), 586.5(ADEFGHILMNPQSTV), N587(-DEGILMPQTV), 587.5(ADEFILMNPQSTVY), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEILMQTVY), Q589(ADEFMNPSTVY), 589.5(ADEFNPSTV), A590(DEGIMPSV), 590.5(ADEFGPST), A591(DEFILMPTVY), 591.5(ACDEFGILMNPQSTVY), T592(-ACDEFGLMNPQSY), 592.5(ACDEFGILMNQSTVWY), A593(-DEFL), 593.5(ACDEFGILMNPQSTVWY), D594(E), 594.5(ACDEFGHILMPQSTVWY), V595(ACEGHILMNPQTW), 595.5(CDEILMTV), N596(CDE), T597(CDEGHILNQSVW), Q598(ACEFHILMSTVY), V600(L), L601(FIMVY), V605(I), W606(F), Q607(M), D608(N), P616(Q), H627(C), P630(ACM), H641(ENT), I646(V), L647(M), N656(A), S658(FNQ), 658.5(NT), T659(NS), T660(H), 660.5(T), F661(Y), S662(M), 662.5(FGSW), A663(CGHIQT), 663.5(DEQ), A664(QT), 664.5(EH), K665(HR), F666(MW), A667(FS), S679(M), V708(W), T716(A), N734(C) W23(EN), P29(AFGIV), P31(NT), P34(A), 34.5(A), E36(V), 36.5(AG), 1.5-fold R37(G), 37.5(ADEGV), H38(ADEGQV), 38.5(AE), R43(T), N66(D), A78(K), Q101(H), K105(N), A117(W), R124(DN), V125(T), L126(FHNY), E127(D), L129(HSY), L131(Y), 132.5(E), 133.5(EN), 134.5(ADEIKNT), P135(ADEFHKNQTW), V136(DEHKPST), 136.5(ADE), 137.5(KT), 138.5(AE), A139(E), 139.5(AEFLNPQSTY), P140(—), 140.5(CDFIPY), G141(-DE), K142(-EGILQSTVY), K143(—), V146(GW), 146.5(DN), H148(-DEQW), 148.5(EY), 149.5(P), P150(FWY), 150.5(ACEFILQVY), 151.5(CPQY), T159(IY), A162(VW), Q164(Y), Q175(Y), 177.5(W), 180.5(S), 181.5(T), V182(W), Q186(VW), G189(DE), 189.5(C), Q190(D), 190.5(EQW), P191(DEFLSWY), 191.5(ILTY), P192(EFT), 192.5(T), T205(H), N270(CD), V372(I), G376(A), N382(D), Q385(W), T410(N), A425(C), I438(AV), Y444(F), L445(C), 445.5(DMNQST), S446(-CNQV), 446.5(ADEFILNPSTVW), R447(-ACDEFGILMNPQSTVWY), 447.5(DEFHILMNPQVWY), T448(-DEFHILMNPQVWY), 448.5(DEP), N449(ACDEFIMP), T450(E), S452(DF), T454(IM), 454.5(DE), T455(DE), 456.5(D), Q457(DE), 457.5(DE), S458(DE), 458.5(DEFW), R459(ACDEGLNPQSTV), L460(ACNSV), Q461(E), S463(N), Q464(AIV), A467(M), D469(E), I470(LM), R471(ACKMQST), D472(Y), Q473(H), C482(M), Y483(V), R484(IKLQ), Q485(C), Q486(DEN), R487(ACDEFGHILMNPQSTVWY), V488(CFMWY), 488.5(D), S489(ADEGMPQW), K490(ACDEFGHILNPQSTVWY), 490.5(DV), T491(DEMQW), 491.5(DEINPQT), S492(E), 492.5(DELQV), A493(EM), 493.5(DN), 494.5(DE), N495(ADEILPQVW), 495.5(DEFWY), N496(ACDEFGHILMVWY), 496.5(DE), N497(-ELV), 497.5(DEMQ), S498(ADEFGLMQWY), 498.5(DFGLMQS), 499.5(ADELMNPQSTV), Y500(-AELNPQSW), 500.5(EGLMNQST), S501(-CDEILMNPTV), 501.5(ACDEGHILMNSTVW), 502.5(ADEFILMSTVWY), T503(PV), 503.5(DEILMNTVW), G504(ACDEFIMVWY), 504.5(CEFILMSTVW), A505(E), 505.5(EIMV), T506(ACILMNV), 506.5(ACV), K507(-CFHILMNQSTVWY), 510.5(K), N511(D), R513(V), L516(ACSTV), V517(I), H526(ACDEGILMNPQSTV), K527(FHR), K532(-AFGILMNPQSTVWY), 532.5(E), F533(ACDEGILMNSTV), 533.5(D), Q536(-CPY), 536.5(CV), S537(-CDGNTY), G538(—), T550(D), N551(E), R566(ACGNSTVW), V571(ES), Q575(GIL), 577.5(E), S578(DEILWY), V579(ACGSY), 579.5(DE), S580(-DENPQ), 580.5(DE), T581(-DENP), 581.5(DEQ), N582(-ACDEFILMPQTVWY), 582.5(GP), L583(DEGIP), 583.5(DPQV), Q584(DEIV), 584.5(ADEFGILMQSTVY), R585(-ADEFGHILMNPQSTVWY), 585.5(ADEFGILMNPQSTVY), G586(-M), 586.5(ADEFGILMNPQSTV), N587(-DEGILMPQTV), 587.5(ADEFILMNQSTVY), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEILMTV), Q589(ADEPSTV), 589.5(ADEFNPTV), A590(DEGIPV), 590.5(ADEGPST), A591(DEFILMTVY), 591.5(ACDEFGILMNPQSTV), T592(-ACDEGNPSY), 592.5(ACDEFGILMNQSTVWY), A593(-DE), 593.5(ACDEFGILMNPQSTVWY), D594(E), 594.5(ADEFHILMPQTVWY), V595(ACEGHLMNPT), 595.5(CDEILV), N596(CDE), T597(DEGHINQVW), Q598(ACEFVY), V600(L), L601(IMY), 662.5(F), 663.5(Q), V708(W) P29(AV), 34.5(A), 37.5(AEGV), H38(AEGV), 38.5(A), Q101(H), R124(N), 2-fold P135(DW), 138.5(E), A139(E), P140(—), K142(I), P150(Y), 150.5(Y), 181.5(T), V182(W), Q186(W), 191.5(Y), N270(D), 445.5(DQ), 446.5(DEN), R447(ACDEILMNPQSTVW), 447.5(DEFHILMNPQWY), T448(- DEFHILPQW), 448.5(DE), N449(ADEF), T450(E), T454(M), 454.5(DE), T455(E), Q457(DE), S458(DE), 458.5(DEW), R459(ACDEPQTV), L460(NS), Q461(E), D469(E), R471(ACKMQST), Y483(V), R484(ILQ), Q485(C), Q486(DEN), R487(ACDEGHILMNPQSTVWY), V488(FMWY), S489(DEGW), K490(ACDEGHLNPQSTVWY), T491(Q), 491.5(DE), S492(E), 492.5(DEQ), A493(E), 493.5(D), 494.5(DE), N495(DEIP), 495.5(DEFWY), N496(DEFGHIMW), 496.5(DE), N497(EV), 497.5(DEMQ), S498(DEFGLMWY), 498.5(DFLMQ), 499.5(ADEMNPQS), Y500(-AELNPQS), 500.5(EGMNQST), S501(ILMNTV), 501.5(ACDEGHILNSTVW), 502.5(ADEFVWY), T503(PV), 503.5(DELTV), G504(DEFMW), 504.5(FIVW), A505(E), 505.5(EI), T506(ILMNV), 506.5(V), K507(-CFHILMNQSTVWY), 510.5(K), R513(V), L516(AST), H526(ACEGIMNQTV), K527(FH), K532(AFGILMPQSTVWY), F533(ACDEGISTV), Q536(-P), 536.5(V), S537(-DNY), G538(—), R566(ACW), V571(E), Q575(IL), 577.5(E), S578(DEIWY), V579(A), 579.5(DE), S580(DENQ), 580.5(DE), T581(-DEN), 581.5(DEQ), N582(-DEFILMPTVWY), L583(D), 583.5(DQ), Q584(DEV), 584.5(ADEFILMQSTVY), R585(-ADFGHILMNPQSTVWY), 585.5(ADEFIMNPQSTVY), 586.5(ADEFGILMPQSTV), N587(DEGILMPQTV), 587.5(ADEFILMQTV), R588(-ACDEFGHILMNPQSTVWY), 588.5(DEILMV), Q589(ADEPSTV), 589.5(ADEFNPTV), A590(DEGP), 590.5(ADEGPS), A591(DEFILMTVY), 591.5(ADEFGILMNPQSTV), T592(DEGY), 592.5(ACDEFGILMNSTVWY), A593(-DE), 593.5(ACDEFGILMNSTWY), 594.5(ADEFILMPQTVWY), V595(AGHLMN), 595.5(CDIV), N596(DE), T597(DEINQVW), Q598(CFVY), L601(Y) 448.5(D), S458(E), R471(AMST), R484(ILQ), Q486(E), 5-fold R487(CGILPQSTVY), V488(Y), K490(E), N495(E), 495.5(D), N496(DEF), 496.5(DE), 497.5(E), S498(D), 498.5(Q), S501(IL), 501.5(DE), K507(CFHILMSTVWY), Q536(—), S537(—), 577.5(E), S578(DE), 579.5(DE), S580(E), 580.5(E), T581(DE), N582(FIPW), Q584(DE), 584.5(D), R585(FIVWY), 585.5(ADNY), 586.5(EILMPV), N587(M), 587.5(DILV), R588(ADFHILMNPSTVWY), 588.5(DE), Q589(DP), 589.5(P), A590(DE), 590.5(DEGP), A591(DE), 591.5(DEFILMV), T592(DE), 592.5(DEFNSW), A593(DE), 593.5(DE), 594.5(DEFV), 595.5(CV), N596(D) R471(AMS), R487(I), K507(CIVWY), S578(D), 580.5(E), T581(DE), 10-fold N582(F), R588(FL), 590.5(D), 591.5(DE), T592(DE), 592.5(D), 593.5(D), 594.5(D)

In one embodiment, a mutation in the viral capsid polypeptide that alters viral tropism as described herein, is combined with a mutation or mutations that increase viral packaging (e.g., any mutation of Table 16).

In another embodiment, a mutation in the viral capsid polypeptide that alters viral tropism as described herein, is combined with a mutation in the region of amino acids selected the group consisting of: 34-38 of SEQ ID NO: 1, 133-152 of SEQ ID NO: 1, 188-194 of SEQ ID NO: 1, and 654-662 of SEQ ID NO: 1 that increases viral packaging.

In yet another embodiment, a mutation in the viral capsid polypeptide that alters viral tropism, as described herein, is combined with any non-positive amino acid insertion, substitution, or deletion in the region of amino acids selected the group consisting of: 442-452 of SEQ ID NO: 1, 489-506 of SEQ ID NO: 1, 541-551 of SEQ ID NO: 1, and 577-596 of SEQ ID NO: 1 that increases viral packaging.

Any region is the packaging heat map that tolerates change, e.g, neutral or positive regions, can potentially tolerate the insertion of additional functional peptide sequences, e.g., an epitope, a tag, a ligand, or other structural sequence that would not necessarily interrupt packaging of a virus.

Methods for Delivering Nucleic Acid

Provide herein is a method of delivering a nucleic acid to a cell comprising contacting a cell with a viral particle comprising any of the viral capsid polypeptides described herein. In one embodiment, the contacting occurs in vitro. In one embodiment, the contacting occurs ex vivo. In another embodiment, the contacting occurs in vivo, e.g., via local or systemic administration.

In one embodiment, the delivering of a nucleic acid is at least 1.1-fold more efficient as compared to a wild-type viral capsid polypeptide. In one embodiment, the the delivering of a nucleic acid is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 5-fold, at least 10-fold, or more, more efficient as compared to a wild-type viral capsid polypeptide. Methods for measuring tropism are described herein above.

In one aspect, a nucleic acid is delivered to a blood cell by contacting a blood cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 1. The cell can be any type blood cell. Exemplary blood cells include, but are not limited to, red blood cells, platelets, neutrophils, eosinophils, basophils, lymphocytes, or monocytes.

In one aspect, a nucleic acid is delivered to a heart cell by contacting a heart cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 2. Exemplary heart cells include, but are not limited to, cardiomyocyte, endocardial cells, or cardiac smooth muscle cells.

In one aspect, a nucleic acid is delivered to a kidney cell by contacting a kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 3. Exemplary kidney cells include, but are not limited to, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, thick ascending limb cells, kidney distal tubule cells, collecting duct principal cells, collecting duct intercalated cells, and interstitial kidney cells.

In one aspect, a nucleic acid is delivered to a liver cell by contacting a liver cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 4. Exemplary liver cells include, but are not limited to, parenchymal cells, non-parenchymal cells, sinusoidal endothelial cells, phagocytic Kupffer cells, hepatic stellate cells, and intrahepatic lymphocytes.

In one aspect, a nucleic acid is delivered to a lung cell by contacting a lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 5. Exemplary lung cells include, but are not limited to, bronchioles, lung epithelial cells, lung smooth muscle cells, and alveoli.

In one aspect, a nucleic acid is delivered to a spleen cell by contacting a spleen cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 6. Exemplary spleen cells include, but are not limited to, splenic endothelial cells and splenic fibroblasts.

Method for Reducing Viral Tropism

One aspect of the technology described herein is a method for reducing tissue tropism of a virus comprising modifying a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1 by introducing a mutation set out in any of Tables 7-9. In one embodiment, the viral tropism for liver, spleen, or blood is reduced.

In one embodiment, the method for reducing viral tropism further comprises any mutation, or combination thereof, as described herein, that increases tropism for a tissue other than the tissue for which the tropism is reduced. For example, a viral capsid polypeptide bearing a mutation which reduces tropism for liver is combined a mutation that increases tropism for kidney. In this example, it is contemplated that this combination of mutations will result in increased tropism for kidney.

Further, one aspect provides a method of increasing delivery of a nucleic acid to a cell of a kidney, heart, or lung, comprising contacting a cell of a kidney, heart, or lung with a viral particle comprising a viral capsid polypeptide comprising a mutation that reduces delivery of a nucleic acid to a cell of a liver, blood, or spleen. In one embodiment, the mutation that reduces delivery of a nucleic acid to a cell of a liver, blood, or spleen is selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

Another aspect provides a method of delivering a nucleic acid to a lung cell comprising contacting a lung cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 5 and a mutation selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

Another aspect provides a method of delivering a nucleic acid to a heart cell, the method comprising contacting a heart cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 2 and a mutation selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

Yet another aspect provides a method of delivering a nucleic acid to a kidney cell comprising contacting a kidney cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 3 and a mutation selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

-   -   1. A viral capsid polypeptide bearing a mutation relative to SEQ         ID NO: 1 (WT AAV2) that alters tissue tropism of a virus         comprising the viral capsid polypeptide, wherein the mutation is         selected from the mutations in any one of Tables 1-9.     -   2. The viral capsid polypeptide of paragraph 1, wherein the         tissue is heart, kidney, liver, lung, spleen, or blood.     -   3. The viral capsid polypeptide of any of the proceeding         paragraphs, wherein the tropism to the tissue is increased.     -   4. The viral capsid polypeptide of any of the proceeding         paragraphs, wherein the tropism to the tissue is decreased.     -   5. A nucleic acid encoding the viral capsid polypeptide of any         of the proceeding paragraphs.     -   6. A viral particle comprising a viral capsid polypeptide of any         of the proceeding paragraphs.     -   7. A viral capsid polypeptide bearing a mutation that         corresponds to a mutation of the polypeptide of SEQ ID NO: 1,         the mutation selected for the group consisting of the mutations         in Tables 1-9 relative to SEQ ID NO: 1.     -   8. A viral capsid polypeptide comprising a region corresponding         to the amino acid sequence of SEQ ID NO: 2, wherein the region         corresponding to the amino acid sequence of SEQ ID NO: 2         comprises a mutation relative to SEQ ID NO: 2 that alters tissue         tropism of a virus comprising the viral capsid polypeptide,         wherein the mutation is selected from the mutations in any one         of Tables 10-15     -   9. The viral capsid polypeptide of any of the proceeding         paragraphs, wherein the tissue is heart, kidney, liver, lung,         spleen, or blood.     -   10. The viral capsid polypeptide of any of the proceeding         paragraphs, wherein the tropism to the tissue is increased.     -   11. The viral capsid polypeptide of any of the proceeding         paragraphs, wherein the tropism to the tissue is decreased.     -   12. A nucleic acid encoding the viral capsid polypeptide of any         of the proceeding paragraphs.     -   13. A viral particle comprising a viral capsid polypeptide of         any of the proceeding paragraphs.     -   14. A method of delivering a nucleic acid to a cell, the method         comprising; contacting the cell with a viral particle comprising         a viral capsid polypeptide of any of the proceeding paragraphs.     -   15. A method of delivering a nucleic acid to a blood cell, the         method comprising; contacting a blood cell with a viral particle         comprising a viral capsid polypeptide comprising a mutation of         Table 1.     -   16. A method of delivering a nucleic acid to a heart cell, the         method comprising; contacting a heart cell with a viral particle         comprising a viral capsid polypeptide comprising a mutation of         Table 2.     -   17. A method of delivering a nucleic acid to a kidney cell, the         method comprising;

contacting a kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation of Table 3.

-   -   18. A method of delivering a nucleic acid to a liver cell, the         method comprising; contacting a liver cell with a viral particle         comprising a viral capsid polypeptide comprising a mutation of         Table 4.     -   19. A method of delivering a nucleic acid to a lung cell, the         method comprising; contacting a lung cell with a viral particle         comprising a viral capsid polypeptide comprising a mutation of         Table 5.     -   20. A method of delivering a nucleic acid to a spleen cell, the         method comprising; contacting a spleen cell with a viral         particle comprising a viral capsid polypeptide comprising a         mutation of Table 6.     -   21. The method of any of the proceeding paragraphs, wherein the         delivering is at least 1.1-fold more efficient as compared to a         wild-type viral capsid polypeptide.     -   22. A method of reducing tissue tropism of a virus comprising a         viral capsid polypeptide corresponding to the viral capsid         polypeptide of SEQ ID NO: 1, the method comprising introducing a         mutation set out in any of Tables 7-9.     -   23. A method of increasing delivery of a nucleic acid to a cell         of a kidney, heart, or lung, the method comprising; contacting a         cell of a kidney, heart, or lung with a viral particle         comprising a viral capsid polypeptide comprising a mutation that         reduces delivery of a nucleic acid to a cell of a liver, blood,         or spleen.     -   24. The method of paragraph 23, wherein the mutation that         reduces delivery of a nucleic acid to a cell of a liver, blood,         or spleen is selected from any of Tables 7-9.     -   25. A method of delivering a nucleic acid to a lung cell, the         method comprising; contacting a lung cell with a viral particle         comprising a viral capsid polypetide comprising a mutation of         Table 5 and a mutation selected from any of Tables 7-9.     -   26. A method of delivering a nucleic acid to a heart cell, the         method comprising; contacting a heart cell with a viral particle         comprising a viral capsid polypetide comprising a mutation of         Table 2 and a mutation selected from any of Tables 7-9.     -   27. A method of delivering a nucleic acid to a kidney cell, the         method comprising;

contacting a kidney cell with a viral particle comprising a viral capsid polypetide comprising a mutation of Table 3 and a mutation selected from any of Tables 7-9.

-   -   28. An AAV2 capsid polypeptide comprising a mutation in the         region of amino acids selected the group consisting of: 440-460         of SEQ ID NO: 1, 475-505 of SEQ ID NO: 1, 518-532 of SEQ ID NO:         1, and 560-590 of SEQ ID NO: 1 that alters tissue tropism of a         virus comprising the viral capsid polypeptide.     -   29. The AAV2 capsid polypeptide of paragraph 28, wherein the         tissue is kidney, liver, or lung.     -   30. The AAV2 capsid polypeptide of paragraph 28, wherein the         tropism is increased.     -   31. An AAV2 capsid polypeptide comprising a mutation in the         region of amino acids 561-588 of SEQ ID NO: 1 that increases         tissue tropism of a virus to the kidney, wherein the mutated         region of amino acid 561-588 of SEQ ID NO: 1 has a sequence         selected from the group consisting of:

(SEQ ID NO: 3) DEEEIATTNPVATEQYGDVSENLMHFQN; (SEQ ID NO: 4) DEEEIRQTNPVATEGYGEVSTNLMHGNK; (SEQ ID NO: 5) DEEEIRTTNPVATEQYGIVnStTNLNEGNR; (SEQ ID NO: 6) DEEEIRTTNPVATECYGSVSTDLQSGNL; (SEQ ID NO: 7) DENEIRTTNPVATEIYGSVSTeNLQNnGdNR; (SEQ ID NO: 8) DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR; (SEQ ID NO: 9) DEEEIRTTNPVATEQYGDVSENLMHFQN.

-   -   32. An AAV2 capsid polypeptide comprising a mutation in the         region of amino acids 561-588 of SEQ ID NO: 1 that increases         tissue tropism of a virus to the liver, wherein the mutated         region of amino acid 561-588 of SEQ ID NO: 1 has a sequence         selected from the group consisting of:

(SEQ ID NO: 10) DEEEIRTTNPVATEQYGVVSDNLQRGNR; (SEQ ID NO: 11) DECEIRTTNPVATEQYGSVGENLQRGNR; (SEQ ID NO: 12) DEEEIRTTNPVATEQYGVVSENLQRGNR; (SEQ ID NO: 13) DESEITTTNPVATEQYGWVSTNQQRGNR; (SEQ ID NO: 14) HELEIATTNPVATEQYGSASTNIQRGNR, (SEQ ID NO: 15) DEEEIATTNPVATEQYGGVSTNLQRGNR.

-   -   33. An AAV2 capsid polypeptide comprising a mutation in the         region of amino acids 561-588 of SEQ ID NO: 1 that increases         tissue tropism of a virus to the lung, wherein the mutated         region of amino acid 561-588 of SEQ ID NO: 1 has a sequence         selected from the group consisting of:

(SEQ ID NO: 16) DEEEIVTTNPVATEQYGNVSTNLQRGNR; (SEQ ID NO: 17) DEDEISTTNPVATEQYGSCSTNLQRGNR; (SEQ ID NO: 18) QEEEIRTTNPVATEQYGSVSTNLQRGDR; (SEQ ID NO: 19) NEEEIRTTNPCATEVYGSVSTNLQRGNR; (SEQ ID NO: 20) DEQEIVTTNPVATEVYGTVSTNLQRGNR.

-   -   34. A method of altering tropism of a virus comprising a capsid         polypeptide corresponding to the polypeptide of SEQ ID NO: 1,         the method comprising introducing a mutation in at least 2         regions selected from the group consisting of: amino acids         440-460 of SEQ ID NO: 1, amino acids 475-505 of SEQ ID NO: 1,         amino acids 518-532 of SEQ ID NO: 1, and amino acids 560-590 of         SEQ ID NO: 1.     -   35. A viral capsid polypeptide bearing a mutation relative to         SEQ ID NO: 1 (WT AAV2) that alters packaging efficiency of a         virus comprising the viral capsid polypeptide, wherein the         mutation is selected from the mutations in Table 16.

REFERENCES

-   1. Adachi, Kei, Enoki, Tatsuji, Kawano, Yasuhiro, Veraz, Michael,     and Nakai Hiroyuki, Drawing a high-resolution functional map of     adeno-associated virus capsid by massively parallel sequencing.     Nature Communications 5, 3075 (2014) -   2. Grimm, D. and Zolotukhin, S. E Pluribus Unum: 50 Years of     Research, Millions of Viruses, and One Goal-Tailored Acceleration of     AAV Evolution. Molecular Therapy 23, 1819-1831 (2015). 

What is claimed is:
 1. A viral capsid polypeptide, wherein the viral capsid polypeptide comprises: a mutation relative to SEQ ID NO: 1 (WT AAV2) that alters tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 1-9; or a region corresponding to the amino acid sequence of SEQ ID NO: 2, wherein the region corresponding to the amino acid sequence of SEQ ID NO: 2 comprises a mutation relative to SEQ ID NO: 2 that alters tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 10-15.
 2. The viral capsid polypeptide of claim 1, wherein the tissue is heart, kidney, liver, lung, spleen, or blood.
 3. The viral capsid polypeptide of claim 1, wherein the tropism to the tissue is increased.
 4. (canceled)
 5. A nucleic acid encoding the viral capsid polypeptide of claim
 1. 6. A viral particle comprising a viral capsid polypeptide claim
 1. 7. (canceled)
 8. (canceled)
 9. The viral capsid polypeptide of claim 1, wherein the tissue is heart, kidney, liver, lung, spleen, or blood. 10.-13. (canceled)
 14. A method of delivering a nucleic acid to a cell, the method comprising; contacting the cell with a viral particle comprising a viral capsid polypeptide of claim
 1. 15. The method of claim 14, wherein the cell is a blood cell and the viral capsid polypeptide comprises a mutation of Table
 1. 16. The method of claim 14, wherein the cell is a heart cell and the viral capsid polypeptide comprises a mutation of Table
 2. 17. The method of claim 14, wherein the cell is a kidney cell and the viral capsid polypeptide comprises a mutation of Table
 3. 18. The method of claim 14, wherein the cell is a liver cell and the viral capsid polypeptide comprises a mutation of Table
 4. 19. The method of claim 14, wherein the cell is a lung cell and the viral capsid polypeptide comprises a mutation of Table
 5. 20. The method of claim 14, wherein the cell is a spleen cell and the viral capsid polypeptide comprises a mutation of Table
 6. 21. (canceled)
 22. A method of reducing tissue tropism of a virus comprising a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1, the method comprising introducing a mutation set out in any of Tables 7-9.
 23. A method of increasing delivery of a nucleic acid to a cell of a kidney, heart, or lung, the method comprising; contacting a cell of a kidney, heart, or lung with a viral particle comprising a viral capsid polypeptide comprising a mutation that reduces delivery of a nucleic acid to a cell of a liver, blood, or spleen
 24. The method of claim 23, wherein the mutation that reduces delivery of a nucleic acid to a cell of a liver, blood, or spleen is selected from any of Tables 7-9.
 25. The method of claim 23, wherein the viral capsid further comprises a mutation of Table 2, 3, or
 5. 26. (canceled)
 27. (canceled)
 28. An AAV2 capsid polypeptide comprising a mutation in the region of amino acids selected the group consisting of: 440-460 of SEQ ID NO: 1, 475-505 of SEQ ID NO: 1, 518-532 of SEQ ID NO: 1, and 560-590 of SEQ ID NO: 1 that alters tissue tropism of a virus comprising the viral capsid polypeptide.
 29. (canceled)
 30. (canceled)
 31. An AAV2 capsid polypeptide, wherein the AAV2 capsid polypeptide comprises: a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the kidney, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of: (SEQ ID NO: 3) DEEEIATTNPVATEQYGDVSENLMHFQN; (SEQ ID NO: 4) DEEEIRQTNPVATEGYGEVSTNLMHGNK; (SEQ ID NO: 5) DEEEIRTTNPVATEQYGIVnStTNLNEGNR; (SEQ ID NO: 6) DEEEIRTTNPVATECYGSVSTDLQSGNL; (SEQ ID NO: 7) DENEIRTTNPVATEIYGSVSTeNLQNnGdNR; (SEQ ID NO: 8) DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR; (SEQ ID NO: 9) DEEEIRTTNPVATEQYGDVSENLMHFQN;

a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the liver, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of: (SEQ ID NO: 10) DEEEIRTTNPVATEQYGVVSDNLQRGNR; (SEQ ID NO: 11) DECEIRTTNPVATEQYGSVGENLQRGNR; (SEQ ID NO: 12) DEEEIRTTNPVATEQYGVVSENLQRGNR; (SEQ ID NO: 13) DESEITTTNPVATEQYGWVSTNQQRGNR; (SEQ ID NO: 14) HELEIATTNPVATEQYGSASTNIQRGNR; (SEQ ID NO: 15) DEEEIATTNPVATEQYGGVSTNLQRGNR;

or a mutation in the region of amino acids 561-588 of SEQ ID NO: 1 that increases tissue tropism of a virus to the lung, wherein the mutated region of amino acid 561-588 of SEQ ID NO: 1 has a sequence selected from the group consisting of: (SEQ ID NO: 16) DEEEIVTTNPVATEQYGNVSTNLQRGNR; (SEQ ID NO: 17) DEDEISTTNPVATEQYGSCSTNLQRGNR; (SEQ ID NO: 18) QEEEIRTTNPVATEQYGSVSTNLQRGDR; (SEQ ID NO: 19) NEEEIRTTNPCATEVYGSVSTNLQRGNR; (SEQ ID NO: 20) DEQEIVTTNPVATEVYGTVSTNLQRGNR.

32.-34. (canceled)
 35. A viral capsid polypeptide bearing a mutation relative to SEQ ID NO: 1 (WT AAV2) that alters packaging efficiency of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in Table
 16. 